Enzymatic nucleic acids containing 5&#39;-and/or 3&#39;-cap structures

ABSTRACT

An enzymatic nucleic acid molecule comprising a 5′- and/or a 3′-cap structure, wherein said structure is not a 5′-5′-linked inverted nucleotide or a 3′-3′-linked inverted nucleotide.

BACKGROUND OF THE INVENTION

This invention relates to chemically synthesized ribozymes, or enzymaticnucleic acid molecules and derivatives thereof.

The following is a brief description of ribozymes. This summary is notmeant to be complete but is provided only for understanding of theinvention that follows. This summary is not an admission that all of thework described below is prior art to the claimed invention.

Ribozymes are nucleic acid molecules having an enzymatic activity whichis able to repeatedly cleave other separate RNA molecules in anucleotide base sequence-specific manner. Such enzymatic RNA moleculescan be targeted to virtually any RNA transcript, and efficient cleavageachieved in vitro (Zaug et al., 324, Nature 429 1986; Kim et al., 84Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 NucleicAcids Research 1371, 1989).

Because of their sequence-specificity, trans-cleaving ribozymes showpromise as therapeutic agents for human disease (Usman & McSwiggen, 1995Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med.Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNAtargets within the background of cellular RNA. Such a cleavage eventrenders the mRNA non-functional and abrogate protein expression fromthat RNA. In this manner, synthesis of a protein associated with adisease state can be selectively inhibited.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. Table I summarizes some of the characteristics of theseribozymes. In general, enzymatic nucleic acids act by first binding to atarget RNA. Such binding occurs through the target binding portion of aenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA will destroy its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over othertechnologies, since the effective concentration of ribozyme necessary toeffect a therapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base-pairing mechanism of binding, but also on the mechanismby which the molecule inhibits the expression of the RNA to which itbinds. That is, the inhibition is caused by cleavage of the RNA targetand so specificity is defined as the ratio of the rate of cleavage ofthe targeted RNA over the rate of cleavage of non-targeted RNA. Thiscleavage mechanism is dependent upon factors additional to thoseinvolved in base-pairing. Thus, it is thought that the specificity ofaction of a ribozyme is greater than that of antisense oligonucleotidebinding the same RNA site.

Chemically-modified ribozymes can be synthesized which are stable inhuman serum for up to 260 hours (Beigelman et al., 1995 supra) andmaintain near wild type (the chemically unmodified equivalent of amodified ribozyme) activity in vitro. A number of laboratories havereported that the enhanced cellular efficacy ofphosphorothioate-substituted antisense molecules. The enhanced efficacyappears to result from either i) increased resistance to 5′-exonucleasedigestion (De Clercq et al., 1970 Virology 42, 421-428; Shaw et al.,1991 Nucleic Acids Res. 19, 747-750), ii) intracellular localization tothe nucleus (Marti et al., 1992 Antisense Res. Dev. 2, 27-39), or iii)sequence-dependent non-specific effects (Gao et al., 1992 Molec.Pharmac. 41, 223-229; Bock et al., 1992 Nature 355, 564-566; and Azad,et al., 1993 Antimicrob. Agents Chemother. 37, 1945-1954) which are notmanifested in nonthioated molecules. Many effects of thioated compoundsare probably due to their inherent tendency to associatenon-specifically with cellular proteins such as the Sp1 transcriptionfactor (Perez et al., 1994 Proc. Natl Acad. Sci. U.S.A. 91, 5957-5961).Chemical modification of enzymatic nucleic acids that provide resistanceto cellular 5′-exonuclease and 3′-exonuclease digestion without reducingthe catalytic activity or cellular efficacy will be important for invitro and in vivo applications of ribozymes.

Modification of oligonucleotides with a 5′-amino group offeredresistance against 5′-exonuclease digestion in vitro (Letsinger &Mungall, 1970 J. Org. Chem. 35, 3800-3803).

Heidenreich et al., 1993 FASEB J. 7, 90 and Lyngstadaas et al., 1995EMBO. J. 14, 5224, mention that hammerhead ribozymes with terminalphosphorothioate linkages can increase resistance against cellularexonucleases.

Seliger et al., Canadian Patent Application No. CA 2,106,819 and Prog.Biotechnol. 1994, 9 (EC B6: Proceedings Of The 6th European Congress OnBiotechnology, 1993, Pt. 2), 681-4 describe “oligoribonucleotide andribozyme analogs with terminal 3′-3′ and/or 5′-5′ internucleotidelinkages”.

SUMMARY OF THE INVENTION

This invention relates to the incorporation of chemical modifications atthe 5′ and/or 3′ ends of nucleic acids, which are particularly usefulfor enzymatic cleavage of RNA or single-stranded DNA. These terminalmodifications are termed as either a 5′-cap or a 3′-cap depending on theterminus that is modified. Certain of these modifications protect theenzymatic nucleic acids from exonuclease degradation. Resistance toexonuclease degradation can increase the half-life of these nucleicacids inside a cell and improve the overall effectiveness of theenzymatic nucleic acids. These terminal modifications can also be usedto facilitate efficient uptake of enzymatic nucleic acids by cells,transport and localization of enzymatic nucleic acids within a cell, andhelp achieve an overall improvement in the efficacy of ribozymes invitro and in vivo.

The term “chemical modification” as used herein refers to any base,sugar and/or phosphate modification that will protect the enzymaticnucleic acids from degradation by nucleases. Non-limiting examples ofsome of the chemical modifications and methods for their synthesis andincorporation in nucleic acids are described in FIGS. 7, 8, 11-16 andinfra.

In a preferred embodiment, chemical modifications of enzymatic nucleicacids are featured that provide resistance to cellular 5′-exonucleaseand/or 3′-exonuclease digestion without reducing the catalytic activityor cellular efficacy of these nucleic acids.

In a second aspect, the invention features enzymatic nucleic acids with5′-end modifications (5′-cap) having the formula:

-   -   wherein, X represents H, alkyl, amino alkyl, hydroxy alkyl,        halo, trihalomethyl [CX₃ (X=Br, Cl, F)], N₃, NH₂, NHR, NR₂ [each        R is independently alkyl (C1-22), acyl (C1-22), or substituted        (with alkyl, amino, alkoxy, halogen, or the like) or        unsubstituted aryl], NO₂, CONH₂, COOR, SH, OR, ONHR, PO₄ ²⁻,        PO₃S²⁻, PO₂S₂ ²⁻, POS₃ ²⁻, PO₃NH²⁻, PO₃NHR⁻, NO₂, CONH₂, COOR, B        represents a natural base or a modified base or H; Y represents        rest of the enzymatic nucleic acid; and R1 represents H,        O-alkyl, C-alkyl, halo, NHR, or OCH₂SCH₃ (methylthiomethyl). The        5′-modified sugar synthesis is as described by Moffatt, in        Nucleoside Analogues:Chemistry, Biology and Medical        Applications, Walker, DeClercq, and Eckstein, Eds,; Plenum        Press: New York, 1979, pp 71 (incorporated by reference herein).

Another preferred embodiment of the invention features enzymatic nucleicacid molecules having a 5′-cap, wherein said cap is selected from butnot limited to, a group comprising, 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; α-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moeity; 5′-5′-inverted abasicmoeity; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moeities (for more details seeBeaucage and lyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

In a third aspect, the invention features enzymatic nucleic acids with3′-end modifications (3′-cap) having the formula:

-   -   wherein, X represents 4′-thio nucleotide, carbocyclic        nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;        α-nucleotides; modified base nucleotide; phosphorodithioate        linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco        nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic        3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide        moeity; 3′-3′-inverted abasic moeity; 3′-2′-inverted nucleotide        moeity; 3′-2′-inverted abasic moeity; 1,4-butanediol;        3′-phosphoramidate; hexylphosphate; aminohexyl phosphate;        3′-phosphate; 3′-phosphorothioate; or bridging or nonbridging        methylphosphonate moeity; B represents a natural base or a        modified base or H; Y represents rest of the enzymatic nucleic        acid; and R1 represents H, O-alkyl, C-alkyl, halo, NHR [R=alkyl        (C1-22), acyl (C1-22), substituted or unsubstituted aryl], or        OCH₂SCH₃ (methylthiomethyl).

In yet another preferred embodiment the invention features enzymaticnucleic acid molecules having a 3′-cap, wherein said cap is selectedfrom but not limited to, a group comprising, 4′-thio nucleotide,carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;α-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moeity; 3′-3′-inverted abasic moeity;3′-2′-inverted nucleotide moeity; 3′-2′-inverted abasic moeity;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or nonbridging methylphosphonate moeity (for more details seeBeaucage and lyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

In a fourth aspect, the invention features enzymatic nucleic acids withboth 5′-cap and a 3′-cap which may be same or different.

The term “nucleotide” is used as recognized in the art to includenatural bases, and modified bases well known in the art. Such bases aregenerally located at the 1′ position of a sugar moiety. Nucleotidegenerally comprise a base, sugar and a phosphate group. The nucleotidecan be unmodified or modified at the sugar, phosphate and/or basemoeity. The term “abasic” or “abasic nucleotide” as used hereinencompasses sugar moieties lacking a base or having other chemicalgroups in place of base at the 1′ position.

By the phrase “enzymatic nucleic acid” is meant a catalyticmodified-nucleotide-containing nucleic acid molecule that hascomplementarity in a substrate binding region to a specified genetarget, and also has an enzymatic activity that specifically cleaves RNAor DNA in that target. That is, the enzymatic nucleic acid is able tointramolecularly or intermolecularly cleave RNA or DNA and therebyinactivate a target RNA or DNA molecule. This complementarity functionsto allow sufficient hybridization of the enzymatic RNA molecule to thetarget RNA or DNA to allow the cleavage to occur. 100% Complementarityis preferred, but complementarity as low as 50-75% may also be useful inthis invention. The nucleic acids may be modified at the base, sugar,and/or phosphate groups. The term enzymatic nucleic acid is usedinterchangeably with phrases such as ribozymes, catalytic RNA, enzymaticRNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,minizyme, leadzyme or DNA enzyme. All of these terminologies describenucleic acid molecules with enzymatic activity.

There are several examples of modified bases as it relates to nucleicacids, is well known in the art and has recently been summarized byLimbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of thenon-limiting examples of base modifications that can be introduced intoenzymatic nucleic acids without significantly effecting their catalyticactivity include, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl,aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines(e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine); Guanosineor adenosine residues may be replaced by diaminopurine residues ineither the core or stems.

There are several examples in the art describing sugar modificationsthat can be introduced into enzymatic nucleic acid molecules withoutsignificantly effecting catalysis and significantly enhancing theirnuclease stability and efficacy. Sugar modification of enzymatic nucleicacid molecules have been extensively described in the art (see Ecksteinet al., International Publication PCT No. WO 92/07065; Perrault et al.Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317;Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman etal. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No.5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702). Suchpublications describe the location of incorporation of modifications andthe like, and are incorporated by reference herein. In view of suchteachings, similar modifications can be used as described herein.

Specifically, an “alkyl” group refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain, and cyclic alkylgroups. Preferably, the alkyl group has 1 to 12 carbons. More preferablyit is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4carbons. The alkyl group may be substituted or unsubstituted. Whensubstituted the substituted group(s) is preferably, hydroxyl, cyano,alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino, or SH. The term also includesalkenyl groups which are unsaturated hydrocarbon groups containing atleast one carbon-carbon double bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkenyl group has 1to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkenyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, —O, ═S, NO₂, halogen, N(CH₃)₂,amino, or SH. The term “alkyl” also includes alkynyl groups which havean unsaturated hydrocarbon group containing at least one carbon-carbontriple bond, including straight-chain, branched-chain, and cyclicgroups. Preferably, the alkynyl group has 1 to 12 carbons. Morepreferably it is a lower alkynyl of from 1 to 7 carbons, more preferably1 to 4 carbons. The alkynyl group may be substituted or unsubstituted.When substituted the substituted group(s) is preferably, hydroxyl,cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group which has at least one ring having a conjugated πelectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above. Carbocyclic arylgroups are groups wherein the ring atoms on the aromatic ring are allcarbon atoms. The carbon atoms are optionally substituted. Heterocyclicaryl groups are groups having from 1 to 3 heteroatoms as ring atoms inthe aromatic ring and the remainder of the ring atoms are carbon atoms.Suitable heteroatoms include oxygen, sulfur, and nitrogen, and includefuranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like, all optionally substituted. An“amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl,alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R iseither alkyl, aryl, alkylaryl or hydrogen.

The 5′-cap and/or 3′-cap derivatives of this invention provide enhancedactivity and stability to the enzymatic nucleic acids containing them.

By “complementarity” is meant a nucleic acid that can form hydrogenbond(s) with other RNA sequence by either traditional Watson-Crick orother non-traditional types (for example, Hoogsteen type) of base-pairedinteractions.

By “bridging” and “nonbridging” are meant to indicate the relativepositions of oxygen atom involved in the formation of standardphosphodiester linkage in a nucleic acid. These backbone oxygen atomscan be readily modified to impart resistance against nuclease digestion.The terms are further defined as follows:

-   -   wherein “x” is bridging oxygen and ‘y’ is nonbridging oxygen.

In preferred embodiments of this invention, the enzymatic nucleic acidmolecule is formed in a hammerhead or hairpin motif, but may also beformed in the motif of a hepatitis delta virus (HDV), group I intron,RNaseP RNA (in association with an RNA guide sequence) or Neurospora VSRNA. Examples of such hammerhead motifs are described by Rossi et al.,1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs byHampel et at, EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929,and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an example ofthe hepatitis delta virus motif is described by Perrotta and Been, 1992Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983Cell 35, 849 and Forster and Altman, 1990 Science 249, 783, NeurosporaVS RNA ribozyme motif is described by Collins (Saville and Collins, 1990Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA88, 8826-8830; Guo and Collins, 1995 EMBO J. 14, 368) and of the Group Iintron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifsare not limiting in the invention and those skilled in the art willrecognize that all that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule.

The invention provides a method for producing a class of enzymaticcleaving agents which exhibit a high degree of specificity for the RNAof a desired target. The enzymatic nucleic acid molecule is preferablytargeted to a highly conserved sequence region of a target such thatspecific treatment of a disease or condition can be provided with asingle enzymatic nucleic acid. Such enzymatic nucleic acid molecules canbe delivered exogenously to specific cells as required. In the preferredhammerhead motif the small size (less than 60 nucleotides, preferablybetween 30-40 nucleotides in length) of the molecule allows the cost oftreatment to be reduced compared to other ribozyme motifs.

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small enzymatic nucleicacid motifs (e.g., of the hammerhead structure) are used for exogenousdelivery. The simple structure of these molecules increases the abilityof the enzymatic nucleic acid to invade targeted regions of the mRNAstructure. Unlike the situation when the hammerhead structure isincluded within longer transcripts, there are no non-enzymatic nucleicacid flanking sequences to interfere with correct folding of theenzymatic nucleic acid structure or with complementary regions.

Therapeutic ribozymes must remain stable within cells until translationof the target mRNA has been inhibited long enough to reduce the levelsof the undesirable protein. This period of time varies between hours todays depending upon the disease state. Clearly, ribozymes must beresistant to nucleases in order to function as effective intracellulartherapeutic agents. Improvements in the chemical synthesis of RNA(Wincott et al., 1995 Nucleic Acids Res. 23, 2677; incorporated byreference herein) have expanded the ability to modify ribozymes toenhance their nuclease stability. The majority of this work has beenperformed using hammerhead ribozymes (reviewed in Usman and McSwiggen,1995 supra) and can be readily extended to other ribozyme motifs.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings:

FIG. 1 is a diagrammatic representation of the hammerhead ribozymedomain known in the art. Stem II can be ≧2 base-pair long. Each N isindependently any base or non-nucleotide as used herein.

FIG. 2 a is a diagrammatic representation of the hammerhead ribozymedomain known in the art; FIG. 2 b is a diagrammatic representation ofthe hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327,596-600) into a substrate and enzyme portion; FIG. 2 c is a similardiagram showing the hammerhead divided by Haseloff and Gerlach (1988,Nature, 334, 585-591) into two portions; and FIG. 2 d is a similardiagram showing the hammerhead divided by Jeffries and Symons (1989,Nucl. Acids. Res., 17, 1371-1371) into two portions.

FIG. 3 is a diagrammatic representation of the general structure of ahairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs(i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided oflength 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 ormore). Helix 2 and helix 5 may be covalently linked by one or more bases(i.e., r is ≧1 base). Helix 1, 4 or 5 may also be extended by 2 or morebase pairs (e.g., 4-20 base pairs) to stabilize the ribozyme structure,and preferably is a protein binding site. In each instance, each N andN′ independently is any normal or modified base and each dash representsa potential base-pairing interaction. These nucleotides may be modifiedat the sugar, base or phosphate. Complete base-pairing is not requiredin the helices, but is preferred. Helix 1 and 4 can be of any size(i.e., o and p is each independently from 0 to any number, e.g., 20) aslong as some base-pairing is maintained. Essential bases are shown asspecific bases in the structure, but those in the art will recognizethat one or more may be modified chemically (abasic, base, sugar and/orphosphate modifications) or replaced with another base withoutsignificant effect. Helix 4 can be formed from two separate molecules,i.e., without a connecting loop. The connecting loop when present may bea ribonucleotide with or without modifications to its base, sugar orphosphate. “q” is ≧2 bases. The connecting loop can also be replacedwith a non-nucleotide linker molecule. H refers to bases A, U, or C. Yrefers to pyrimidine bases. “______” refers to a covalent bond.

FIG. 4 is a representation of the general structure of the hepatitisdelta virus ribozyme domain known in the art. In each instance, each Nand N′ independently is any normal or modified base and each dashrepresents a potential base-pairing interaction. These nucleotides maybe modified at the sugar, base or phosphate.

FIG. 5 is a representation of the general structure of the self-cleavingVS RNA ribozyme domain.

FIG. 6 is a diagrammatic representation of a hammerheadribozyme-substrate complex. The ribozyme is targeted against site 575within c-myb RNA. Lowercase alphabets indicate 2′-O-methyl substitution;uppercase alphabets indicate ribonucleotides; Arrow incates the site ofRNA cleavage; u₄ and u₇, represent modification with 2′-amino group; Xand Z, represent 5′- and 3′-caps which may be same or different.

FIG. 7 A) is a general formula for 5′-end modifications. B) chemicalstructures of a few of the 5′-end modifications. C) diagrammaticrepresentation of a 5′-5′-inverted abasic moiety.

FIG. 8A) diagrammatic representation of 5′-phosphoramidate and5′-phosphorothioate linkages; B) a synthesis scheme for5′-amino-5′-deoxy-2′-O-methyl uridine and guanosine phosphoramidites; C)a synthesis scheme for 5′-amino-5′-deoxy-2′-O-methyl adenosinephosphoramidites; D) a synthesis scheme for5′-deoxy-5′-mercapto-2′-O-methyl uridine and cytidine phosphoramidites.

FIG. 9 shows ribozyme-mediated inhibition of smooth muscle cellproliferation. The hammerhead (HH) ribozymes, targeted to site 575within c-myb RNA, as shown in FIG. 6, were chemically modified such thatthe ribozyme consists of ribose residues at five positions; U4 and U7positions contain 2′-NH₂ modifications, the remaining nucleotidepositions contain 2′-O-methyl substitutions. Additionally, the 5′-end ofthe ribozyme contains 5′-amino modification and the 3′ end of theribozyme contains a 3′-3′ linked inverted abasic deoxyribose (designatedas 3′-iH). Inactive ribozyme (5′-amino Inactive RZ) with G5 to U and A14to U substitution was synthesized and used as a negative control.

FIG. 10 shows ribozyme-mediated inhibition of smooth muscle cellproliferation. The hammerhead (HH) ribozymes, targeted to site 575within c-myb RNA, as shown in FIG. 6, were chemically modified such thatthe ribozyme consists of ribose residues at five positions; U4 positioncontains 2′-C-allyl modification, the remaining nucleotide positionscontain 2′-O-methyl substitutions. Additionally, the 5′-end of theribozyme contains amino modification and the 3′ end of the ribozymecontains a 3′-3′ linked inverted abasic deoxyribose (designated as3′-iH). Inactive ribozyme (5′-amino Inactive RZ) with G5 to U and A14 toU substitution was synthesized and used as a negative control.

FIG. 11 A) chemical structures of a few of the 3′-end modifications. B)diagrammatic representation of a few 3′-end mofication linkages.

FIG. 12 is a synthesis scheme for phosphorodithioate linkages.

FIG. 13 is a synthesis scheme for 3′-2′-inverted nucleoside or an abasicnucleoside linkages. Compound 2 can be reacted with compound 3 to yieldeither a 3′-2′-inverted nucleotide linkage as shown in FIG. 11B, infra,or a 3′-2′-inverted abasic ribose, deoxyribose or variations thereof(see FIG. 11B).

FIG. 14 is a synthesis scheme for carbocyclic nucleosidephosphoramidite.

FIG. 15 is a synthesis scheme for alpha nucleoside phosphoramidite.

FIG. 16 is a synthesis scheme for 1-(β-D-erythrofuranosyl) nucleosidephosphoramidite.

FIG. 17 is a synthesis scheme for inverted deoxyabasic 5′-O-succinateand 5′-O-phosphoramidite.

FIG. 18 is a graphical representation of RNA cleavage reaction catalyzedby hammerhead ribozymes containing either one or two 5′-terminalphosphorodithioate modifications. Ribozyme 0.4654/1 5′-dithio,represents a hammerhead ribozyme targeted to c-myb site 575 as shown inFIG. 6, and were chemically modified such that the ribozyme consists ofribose residues at five positions; U4 position contains 2′-C-allylmodification, the remaining nucleotide positions contain 2′-O-methylsubstitutions. Additionally, the 5′-end of the ribozyme contains onephosphorodithioate modification and the 3′ end of the ribozyme containsa 3′-3′ linked inverted abasic deoxyribose (designated as 3′-iH).Ribozyme 0.4657/2 5′-dithio, represents a hammerhead ribozyme targetedto c-myb site 575 as shown in FIG. 6, and were chemically modified suchthat the ribozyme consists of ribose residues at five positions; U4position contains 2′-C-allyl modification, the remaining nucleotidepositions contain 2′-O-methyl substitutions. Additionally, the 5′-end ofthe ribozyme contains two phosphorodithioate modification and the 3′ endof the ribozyme contains a 3′-3′ linked inverted abasic deoxyribose(designated as 3′-iH).

Nucleotides and Nucleosides

Applicant has found that chemical modifications of this invention areparticulary useful for enzymatic nucleic acid molecule stabilization.Thus, below is provided examples of one such molecule, a hammerheadribozyme. Those in the art will recognize that equivalent procedures canbe used to make other enzymatic nucleic acid molecules having a 5′-and/or 3′-cap structure. Specifically, FIGS. 1 and 6 show base numberingof a hammerhead motif in which the numbering of various nucleotides in ahammerhead ribozyme is provided. This is not to be taken as anindication that the Figure is prior art to the pending claims, or thatthe art discussed is prior art to those claims.

EXAMPLES

The following are non-limiting examples showing the synthesis andactivity of enzymatic nucleic acids containing 5′- and/or 3′-capmodifications and the synthesis of monomer phosphoramidites.

Example 1 Synthesis of Enzymatic Nucleic Acids Containing 5′- and/or3′-Cap Structures

The method of synthesis follows the procedure for normal RNA synthesisas described in Usman, N.; Ogilvie, K. K.; Jiang, M.-Y.; Cedergren, R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854; Scaringe, S. A.; Franklyn,C.; Usman, N. Nucleic Acids Res. 1990, 18, 5433-5441; and Wincott etal., 1995, Nucleic Acids Res. 23, 2677 (all of these references areincorporated by reference herein in their entirety) and makes use ofcommon nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.Phosphoramidites of the 5′-cap and/or 3′-cap structures selected fromthose described and illustrated in FIGS. 7-8 and 11-16 may beincorporated not only into hammerhead ribozymes, but also into hairpin,hepatitis delta virus, VS RNA, RNase P ribozyme, Group I or Group IIintron catalytic nucleic acids. They are, therefore, of general use inany enzymatic nucleic acid structure.

Example 2 Incorporation of 5′-Amino- and5′-Mercapto-5′-Deoxy-2′-O-Methyl Nucleosides into Hammerhead Ribozymes

Non-chiral phosphoramidate and phosphorothioate linkages (FIG. 8) forincorporation at the 5′-end of a hammerhead ribozyme are describedinfra. These linkages are electronically and sterically similar to theirnatural congener and introduction of a single 3′-O—P(O)(O⁻)—NH-5′ or3-O—P(O)(O⁻)—S-5′ link at the 5′-end of the ribozyme has little effecton its hybridization to a substrate and/or ribozyme cleavage activity.Letsinger and Mungall, J. Org. Chem. 1970, 35, 3800-3803, reported thesynthesis of a thymidine dimer and trimer possessing internucleotidephosphoramidate bonds 3′-O—P(O)(O⁻)—NH-5′ which were stable in neutraland alkaline conditions and showed increased stability againstexonucleases. The terminal 5′-amino group of a thymidine dimer was foundto efficiently inhibit the action of spleen phosphodiesterase. It isalso reported that introduction of a phosphoramidate 3′-NH—P(O)(O⁻)—O-5′leads to enhancement in stability of the heteroduplex (Gryaznov andLetsinger, Nucleic Acids Res. 1992, 20, 3403-3409). While studies of3′-S-modified oligodeoxynucleotides demonstrated complete resistance tocleavage by EcoRV, there are no related studies on 5′-S-modifiedoligonucleotides (Vyle et al., Biochemistry 1992, 31, 3012-3018).Although there is interest in the synthesis, chemical and biologicalproperties of oligonucleotides with bridging 5′-N (Letsinger et al.,supra; Mag and Engels, Tetrahedron 1994, 50, 10225-10234; Gryaznov andSokolova, Tetrahedron Lett. 1990, 31, 3205-3208; Letsinger et al.,Nucleic Acids Res. 1976, 3, 1053-1063; Mag, and Engels, Nucleosides &Nucleotides 1988, 7, 725-728) and 5′-S (Sund and Chattopadhyaya,Tetrahedron 1989, 45, 7523-7544; Chladek amnd Nagyvary, Amer. Chem. Soc.1972, 94, 2079-2085; Cook, J. Amer. Chem. Soc. 1970, 92, 190-195; Liuand Reese, Tetrahedron Lett. 1995, 36, 3413-3416) substitutions as wellas 3′-N (Mag et al., Tetrahedron Lett. 1992, 33, 7319-7322; Zielinskiand Orgel, Nucleic Acids Res. 1987, 15, 1699-1715) and 3′-S (Cosstickand Vyle, Nucleic Acids Res. 1990, 18, 829-835; Li et al., Tetrahedron1992, 48, 2729-2738; and J. Chem. Soc. Perkin I 1994, 2123-2129; Liu andReese, Tetrahedron Lett. 1996, 37, 925-928) modified oligonucleotides,there are few reports (Bannwarth, Helv. Chim. Acta 1988, 71, 1517-1427;Mag and Engels, Nucleic Acids Res. 1989, 17, 5973-5988; Mag et al.,Nucleic Acids Res. 1991, 19, 1437-1441; Chen et al., Nucleic Acids Res.1995, 23, 2661-2668; Cosstick and Vyle Tetrahedron Lett. 1989, 30,4693-4696) of the step-by-step elongation on solid support using 5′- or3′-N(S)-modified nucleotide monomers.

Because of the different chemical nature of N—R and S—R bonds comparedto O—R bonds there is a requirement for introduction of specialprotecting groups for amino and thiol functions and special conditionsfor their cleavage, considerably different from those routinely used ina solid phase nucleic acid synthesis, but still compatible with solidphase phosphoramidite chemistry. Also, optimization of the syntheticcycle for the introduction of the modified monomers is usuallynecessary.

Based on previous investigations in the 2′-deoxy series (Mag et al.,1989 and 1991 supra) we have chosen 4-methoxytrityl (MMTr) group for theprotection of the 5′-amino function while the trityl (Tr) group was usedfor the protection of the 5′-mercapto functionality in modifiedmonomers.

The synthesis of 5′-amino-5′-deoxy-2′-O-methyl-uridine, guanosine andadenosine 3′-phosphoramidites 5, 11 and 20 (FIGS. 8B and 8C), as well as5′-mercapto-5′-deoxy-2′-O-methyl-uridine and cytidine 3′-phosphoramidite23 and 28 (FIG. 8D) and their incorporation into ribozymes are describedinfra. Extensive modification of hammerhead ribozyme with2′-O-Me-nucleosides resulted in a catalytic motif with almost wild typecleavage activity and considerably improved nuclease stability hasrecently been described (Beigelman et al., J. Biol. Chem. 1995, 270,25702-25708). Another reason for using 2′-O-methyl modified nucleotidesis to prevent degradation of oligonucleotides by attack of the freeneighboring 2′-hydroxyl on the phosphorus during deprotection, a welldocumented event in the case of 5′-S-modified ribonucleoside dimers.

Materials and Methods

General Methods

2′-O-Methyluridine, N²-isobutyryl-2′-O-methylguanosine and5′-O-(4,4′-dimethoxytrityl)-N⁶-benzoyl-2′-O-methyladenosine wereobtained from ChemGenes Corporation (Waltham, Mass.). All NMR spectrawere recorded on a Varian Gemini 400 spectrometer operating at 400.075MHz for proton and 161.947 MHz for phosphorus. Chemical shifts in ppmrefer to TMS and H₃PO₄, respectively. The solvent was CDCl₃ if notstated otherwise. The standard work up consisted of partitioning of theresidue after removal of solvents between 5% aqueous NaHCO₃ and CH₂Cl₂followed by washing of the organic layer with brine, drying over Na₂SO₄and removal of solvents in vacuo. Analytical thin-layer chromatography(TLC) was performed with Merck Art. 5554 Kieselgel 60 F₂₅₄ plates andcolumn chromatography using Merck 0.040-0.063 mm Silica gel 60. Meltingtemperatures were determined on the Electrothermal Model IA 9200apparatus and are uncorrected.

The general procedures for RNA synthesis and deprotection have beendescribed previously (Wincott et al., supra, incorporated by referenceherein in its entirety) Syntheses were conducted on a 394 (ABI)synthesizer using a modified 2.5 μmol scale protocol with a 5 mincoupling step for 2′-O-TBDMSi protected nucleotides and 2.5 min couplingstep for 2′-O-methyl nucleotides. A 6.5-fold excess of a 0.1 M solutionphosphoramidite and a 24-fold excess of S-ethyl tetrazole relative topolymer-bound 5′-hydroxyl was used in each coupling cycle.

All analytical HPLC analyses were performed on a Hewlett Packard 1090HPLC with a Dionex NucleoPac® PA-100 column, 4×250 mm, at 50° C., asreported (Wincott et al., supra).

CGE analyses were performed on a Hewlett Packard ^(3D)CE with a J & WμPAGE™-5 (5% T, 5% C) polyacrylamide gel-filled column, 75 μm I.D.×75cm, 50 cm effective length, 100 mM Tris-Borate, 7 M Urea, pH=8.3, and J& W μPAGE™ Buffer (100 mM Tris-Borate, 7 M Urea, pH=8.3). Samples wereelectrokinetically injected using −13 kV for 3-10 sec, run at −13 kV anddetected at 260 nm.

MALDI-TOF mass spectra were determined on a PerSeptive BiosystemsVoyager spectrometer.

Synthesis of Monomer Building Blocks

Referring to FIG. 8B, 5′-Azido-5′-deoxy-2′-O-methyluridine (2) wassynthesized from 2′-O-methyluridine (1) in 79% yield (white foam)according to the procedure of Yamamoto et al., J. Chem. Soc. Perkin I1978, 306-310 (incorporated by reference herin in its entirety), for thepreparation of 5′-azido-5′-deoxythymidine, ¹H NMR δ 9.16 (br s, 1H, NH),7.70 (d, J_(6,5)=8.2, 1H, H6), 5.97 (d, J_(1′,2′)=2.2, 1H, H1′), 5.87(d, J_(5,6)=8.2, 1H, H5), 4.21 (m, 1H,H3′), 4.08 (m, 1H, H2′), 3.92 (dd,J_(5′,4′)=2.2, J_(5′,5″)=13.4, 1H, H5′), 3.87 (dd, J_(4′,5′)=2.2,J_(4′,3′)=5.6, 1H, H4′), 3.82 (dd, J_(5″,4′)=3.3, J_(5″,5′)=13.4, 1H,H5″), 3.67 (s, 3H, OMe).

5′-Amino-5′-deoxy-2′-O-methyluridine (3) (FIG. 8B) was synthesized from2 according to a modification of the procedure of Mag and Engels,Nucleic Acids Res. 1989, 17, 5973-5988 (incorporated by reference herinin its entirety), for the preparation of 5′-amino-5′-deoxythymidine: 2(680 mg, 2.27 mmol) was dissolved in dry pyridine (5 mL) andtriphenylphosphine (Ph₃P) (890 mg, 3.39 mmol) was added. The mixture wasstirred for 2 h at rt at which time all the starting material hadreacted. Concentrated NH₄OH (2 mL) was then added and the mixturestirred at rt for 2 h. Solvents were removed at reduced pressure, waterwas added (20 mL) and precipitate removed by filtration. The filtratewas extracted with benzene and ether and then evaporated to dryness. Theresidue was dissolved in isopropanol from which the amorphous solidprecipitated on cooling (480 mg, 82%), ¹H NMR (dmso-d₆) δ 8.01 (d,J_(6,5)=8.1, 1H, H6), 5.90 (d, J_(1′,2′)=5.2, 1H, H1′), 5.71 (d,J_(5,6)=8.1, 1H, H5), 4.16 (app t, J_(3′,4′)=5.0, 1H, H3′), 3.91 (app t,J_(2′,1′)=5.2, 1H, H2′), 3.84 (q, J_(4′,3′)=5.0, 1H, H4′), 3.43 (s, 3H,OMe), 2.88 (dd, J_(5′,4′)=4.5, J_(5′,5″)=13.7, 1H, H5′), 2.83 (dd,J_(5″,4′)=5.0, J_(5″,5′)=13.7, 1H, H5″).

5′-N-(4-Methoxytrityl)amino-5′-deoxy-2′-O-methyluridine (4) (FIG. 8B)was synthesized from 3 using 4-methoxytrityl chloride/DMAP/Et₃N/Pyr in63% yield according to the procedure of Mag and Engels, Nucleic AcidsRes. 1989, 17, 5973-5988, and is incorporated by reference herin in itsentirety. ¹H NMR δ 8.25 (br s, 1H, NH), 7.54-6.88 (m, 15H, aromatic,H6), 5.96 (s, 1H, H1′), 5.70 (d, J_(5,6)=7.9, 1H, H5), 4.13 (m, 1H,H3′), 4.01 (m, 1H, H2′), 3.86 (s, 3H, TrOMe), 3.77 (m, 1H, H4′), 3.69(s, 3H, OMe), 2.82 (dd, J_(5′,4)=2.9, J_(5′,5″)=12.9, 1H, H5′), 2.66 (d,J_(NH,5′)=8.8, 1H, 5′NH), 2.42 (dd, J_(5″,4′)=6.8, J_(5″,5′)=12.9, 1H,H5″).

5′-N-(4-Methoxytrityl)amino-5′-deoxy-2′-O-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)(5). (see FIG. 8B) To the solution of 4 (520 mg, 0.98 mmol) andN,N-diisopropylethylamine (DIPEA) (0.34 mL, 1.95 mmol) in CH₂Cl₂ (10 mL)under argon was added 2-cyanoethyl N,N-diisopropylchlorophosphoramidite(0.30 mL, 1.34 mmol) was added dropwise, stirring was continued for 3 hat rt. The reaction mixture was then cooled to 0° C., dry MeOH (3 mL)was added and stirring continued for 5 min. The mixture was evaporatedto dryness in vacuo (40° C. bath temp) and the residue chromatographedon a silica gel column using 20-70% gradient EtOAc in hexane (1% Et₃N)to afford 5 as a colorless foam (0.60 g, 83%), ³¹P NMR δ 148.97 (s) and148.67 (s).

5′-O-p-Toluenesulfonyl-N²-isobutyryl-2′-O-methylguanosine (7). (see FIG.8B) N²-Isobutyryl-2′-O-methylguanosine (6) (Inoue et al., Nucleic AcidsRes. 1987, 15, 6131-6148, and is incorporated by reference herin in itsentirety) (1.6 g, 4.36 mmol) was dissolved in dry pyridine (25 mL) andthe solution was cooled to 0° C. while protected from moisture.p-Toluenesulfonyl chloride (1.0 g, 5.23 mmol) was added and the reactionmixture was left at 0-3° C. for 48 h. MeOH (10 mL) was added and themixture evaporated to a syrup. After standard work up and columnchromatography using 1-2% MeOH in CH₂Cl₂, 7 was obtained as a colorlessfoam, 1.06 g (47%), ¹H NMR δ 12.25 (br s, 1H, NH), 9.55 (br s, 1H, NH),7.83 (d, J_(H,H)=8.3, 2H, Ts), 7.78 (s, 1H, H8), 7.42 (d, J_(H,H)=8.3,2H, Ts), 5.83 (d, J_(1′,2′)=6.2, 1H, H1′), 4.82 (app t, J_(2′,3′)=5.7,1H, H2′), 4.64 (m, 1H, H3′), 4.37 (dd, J_(5′,4′)=2.2, J_(5′,5″)=10.3,1H, H5′), 5.23 (dd, J_(4′,5″)=2.9, J_(4′,3′)=5.2, 1H, H4′), 4.29 (dd,J_(5″,4′)=2.9, J_(5″,5′)=10.3, 1H, H5″), 3.47 (s, 3H, OMe), 2.76 (m, 1H,CH(CH₃)₂), 2.51 (s, 3H, Ts-Me), 1.29 (m, 6H, 2×Me).

The 3′,5′-Di-O-p-toluenesulfonyl derivative was also isolated (0.45 g,15%) from the reaction mixture along with 20% of the unreacted startingmaterial.

As shown in FIG. 8B 5′-Azido-5′-deoxy-N²-isobutyryl-2′-O-methylguanosine(8). 7 (780 mg, 1.5 mmol) was dissolved in dry DMSO (7 mL) and LiN₃ (370mg, 7.56 mmol) was added under argon. The mixture was heated at 50° C.for 16 h and then evaporated to a syrup (oil pump, 50° C.). The residuewas partitioned between water (30 mL) and EtOAc (30 mL). The aqueouslayer was extracted with EtOAc (4×20 mL), organic layers combined, dried(Na₂SO₄) and evaporated to dryness. Flash column silica gelchromatography using 2-25% MeOH in CH₂Cl₂ afforded 8, 430 mg (78%), mp107-109° C. (H₂O), ¹H NMR (dmso-d₆) δ 12.17 (br s, 1H, N H), 11.68 (brs, 1H, NH), 8.36 (s, 1H, H8), 6.01 (d, J_(1′,2′)=6.1, 1H, H1′), 5.52 (d,J_(OH,3′)=5.1, 1H, 3′OH), 4.47 (app t, J_(2′,3′)=5.5, 1H, H2′), 4.37 (m,1H, H3′), 4.12 (m, 1H, H4′), 3.75 (dd, J_(5′,4′)=6.8, J_(5′,5″)=13.2,1H, H5′), 3.65 (dd, J_(5″,4′)=4.2, J_(5″,5′)=13.2, 1H, H5″), 3.43 (s,3H, OMe), 2.86 (m, 1H, CH(CH₃)₂), 1.22 (s, 3H, Me), 1.20 (s, 3H, Me).

5′-Amino-5′-deoxy-N²-isobutyryl-2′-O-methylguanosine (9) (FIG. 8B) Tothe solution of 8 (350 mg, 0.95 mmol) in 96% EtOH (30 mL) 10% Pd/Ccatalyst (60 mg) was added. The mixture was hydrogenated under 35 psi ofH₂ for 24 h. More EtOH was added and heated to get the partlycrystallized product completely into solution. Then the catalyst wasfiltered off. On cooling, crystals formed which were filtered off anddried to give 260 mg in two crops (80%), mp 197-199° C. ¹H NMR (D₂O) δ8.16 (s, 1H, H8), 6.15 (d, J_(1′,2′)=4.6, 1H, H1′), 4.66 (app t,J_(3′,2′)=5.4, J_(3′,4′)=5.4, 1H, H3′), 4.57 (app t, J_(2′,1′)=4.6,J_(2′,3′)=5.4, 1H, H2′), 4.34 (m, 1H, H4′), 3.50 (s, 3H, OMe), 3.49 (m,2H, H5′,H5″), 2.82 (m, 1H, CH(CH₃)₂), 1.26 (s, 3H, Me), 1.24 (s, 3H,Me).

5′-N-(4-Methoxytrityl)amino-5′-deoxy-N²-isobutylyl-2′-O-methylguanosine(10) was synthesized from 9 using 4-methoxytrityl chloride/DMAP/Et₃N/Pyr(FIG. 8B) according to the procedure of Mag and Engels, supra, in 80%yield. ¹H NMR δ 12.11 (br s, 1H, NH), 7.95 (br s, 1H, NH), 7.70 (s, 1H,H8), 7.53-6.86 (m, 14H, aromatic), 5.92 (d, J_(1′,2′)=4.9, 1H, H1′),4.55 (app t, J_(3′,4′)=5.0, 1H, H3′), 4.35 (app t, J_(2′,1′)=4.9, 1H,H2′), 3.84 (s, 3H, Tr-OMe), 3.55 (s, 3H, OMe), 2.82 (br s, 1H, 3′OH),2.78 (dd, J_(5′,4′)=3.0, J_(5′,5″)=12.4, 1H, H5′), 2.65 (br s, 1H, NH),2.43 (dd, J_(5″,4′)=5.4, J_(5″,5′)=12.4, 1H, H5″), 1.09 (m, 6H, 2×Me).

5′-N-(4-Methoxytrityl)amino-5′-deoxy-N²-isobutyryl-2′-O-methylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)(11). Using the same procedure as for the preparation of 5,phosphoramidite 11 was obtained (FIG. 8B) as a colorless foam in 80%yield after column chromatography using 1% EtOH in CH₂Cl₂ (1% Et₃N), ³¹PNMR δ 148.74 (s) and 148.06 (s).

Referring to FIG. 8C,3′-O-t-Butyldiphenylsilyl-N⁶-benzoyl-2′-O-methyladenosine (13).5′-O(4,4′-Dimethoxytrityl)-N⁶-benzoyl-2′-O-methyladenosine 12 (5 g, 7.3mmol) was dissolved in DMF (20 mL) and imidazole (1.5 g, 22 mmol) andt-butyldiphenylsilyl chloride (2.8 mL, 10.8 mmol) were added. Themixture was stirred at rt overnight. Methanol (10 mL) was added and thesolution evaporated to a syrup. After standard work up the resultingsyrup was dissolved in CH₂Cl₂ (100 mL) and cooled in an ice-bath. 3% TFAin CH₂Cl₂ (v/v, 100 mL) was added and the mixture was stirred at 0° C.for 10 min. Methanol (20 mL) and toluene (50 mL) were added and thesolution concentrated to a syrup in vacuo (40° C.). The residue wascoevaporated twice with toluene and then purified by columnchromatography using 1-5% MeOH in CH₂Cl₂ for elution to yield 13 as awhite foam (4.3 g, 95% yield), ¹H NMR δ 8.98 (br s, 1H, NH), 8.73 (s,1H, H2), 8.13 (s, 1H, H8), 8.02-7.39 (m, 15H, 3×Ph), 6.06 (d,J_(1′,2′)=7.4, 1H, H1′), 5.86 (d, J_(OH,5′)=10.2, 1H, 5′OH), 4.55 (m,2H, H2′,H3′), 4.20 (br s, 1H, H4′), 3.70 (d, J_(5″,5′)=12.9, 1H, H5′),3.14 (d, J_(5″,5′)=12.9, 1H, H5″), 3.10 (s, 3H, OMe), 1.15 (s, 9H,t-Bu).

5-O-(4-Nitrobenzenesulfonyl)-3′-O-t-butyldiphenylsilyl-N⁶-benzoyl-2′-O-methyladenosine(14) and5′-chloro-5′deoxy-3′-O-t-butyldiphenylsilyl-N⁶-benzoyl-2′-O-methyladenosine(15). (see FIG. 8C). To a solution of 13 (4.3 g, 6.9 mmol) in drypyridine (70 mL) was added 4-nitrobenzenesulfonyl chloride (2.47 g, 11mmol) and the solution was left at rt overnight. Water (2 mL) was addedand the solution concentrated to a syrup in vacuo. After standard workup the reaction mixture was purified by column chromatography using 1-5%gradient MeOH in CH₂Cl₂ to yield 4.7 g of the inseparable mixture of 14and 15 in 2:1 ratio, ¹H NMR for 14 δ 8.89 (br s, 1H, NH), 8.58 (s, 1H,H2), 8.16-7.36 (m, 20H, H8, aromatic), 6.00 (d, J_(1′,2′)=3.8, 1H, H1′),4.56 (app t, J_(3′,4′)=5.1, 1H, H3′), 4.33 (m, 1H, H4′), 4.27 (dd,J_(5′,4′)=2.8, J_(5′,5″)=11.2, 1H, H5′), 4.14 (dd, J_(5″,4′)=5.3,J_(5″,5′)=11.2, 1H, H5″), 4.09 (app t, J_(2′,1′)=3.8, 1H, H2′), 3.20 (s,3H, OMe), 1.11 (s, 9H, t-Bu), ¹H NMR for 15 δ 8.92 (br s, 1H, NH), 8.71(s, 1H, H2), 8.16-7.36 (m, 20H, H8, aromatic), 6.15 (d, J_(1′,2′)=3.9,1H, H1′), 4.51 (app t, J_(3′,4′)=5.1, 1H, H3′), 4.42 (m, 1H, H4′), 4.06(app t, J_(2′,1′)=3.9, 1H, H2′), 3.82 (dd, J_(5′,4′)=4.3,J_(5′,5″)=12.1, 1H, H5′), 3.54 (dd, J_(5″,4′)=3.9, J_(5″,5′)=12.1, 1H,H5″), 3.25 (s, 3H, OMe), 1.13 (s, 9H, t-Bu).

5′-Azido-5′-deoxy-3′-O-t-butyldiphenylsilyl-N⁶-benzoyl-2′-O-methyladenosine(16). (FIG. 8C) The above mixture of 14 and 15 (3.9 g) was dissolved indry DMSO (30 mL) and LiN₃ (1.18 g, 24 mmol) was added. The reactionmixture was stirred at 80° C. overnight, then concentrated in vacuo (oilpump). After standard work up and column chromatography using 1-2%gradient MeOH in CH₂Cl₂ 16 was obtained as a colorless foam (2.55 g), ¹HNMR δ 8.92 (br s, 1H, NH), 8.72 (s, 1H, H2), 8.15 (s, 1H, H8), 8.02-7.36(m, 15H, 3×Ph), 6.14 (d, J_(1′,2′)=3.4, 1H, H1′), 4.44 (app t,J_(3′,4′)=5.1, 1H, H3′), 4.27 (m, 1H, H4′), 4.01 (app t, J_(2′,1′)=3.4,J_(2′,3′)=4.9, 1H, H2′), 3.53 (dd, J_(5′,4′)=3.2, J_(5′,5″)=13.3, 1H,H5′), 3.37 (dd, J_(5″,4′)=4.5, J_(5″,5′)=13.3, 1H, H5″), 3.29 (s, 3H,OMe), 1.13 (s, 9H, t-Bu).

5′-Amino-5′-deoxy-3′-O-t-butyldiphenylsilyl-N⁶-benzoyl-2′-O-methyladenosine(17). Using the same procedure (FIG. 8B) as for the preparationguanosine analog 9, 16 (2.5 g, 3.9 mmol) was converted into 17 (2.25 g,94%) which resisted crystallization and was used crude in the next step,¹H NMR δ 8.90 (br s, 1H, NH), 8.72 (s, 1H, H2), 8.23 (s, 1H, H8),8.02-7.36 (m, 15H, aromatic), 6.13 (d, J_(1′,2′)=4.4, 1H, H1′), 4.72(app t, J_(2′,1′)=4.4, J_(2′,3′)=5.0, 1H, H2′), 4.17 (m, 2H, H3′, H4′),3.27 (s, 3H, OMe), 2.88 (dd, J_(5′,4′)=3.2, J_(5′,5″)=13.8, 1H, H5′),2.65 (dd, J_(5″,4′)=5.0, J_(5″,5′)=13.8, 1H, H5″), 1.12 (s, 9H, t-Bu).

5′-N-(4-Methoxytrityl)amino-5′-deoxy-3′-O-t-butyldiphenylsilyl-N⁶-benzoyl-2′-O-methyladenosine(18). (FIG. 8C) Using the same procedure as for the preparation of 10,17 was converted into 18, which was then purified by columnchromatography using 1-2% MeOH gradient in CH₂Cl₂, (2.37 g, 76%) as acolorless foam, ¹H NMR δ 8.90 (br s, 1H, NH), 8.02 (s, 1H, H2), 7.95 (s,1H, H8), 8.00-6.71 (m, 29H, 3×Ph), 6.04 (d, J_(1′,2′)=6.4, 1H, H1′),4.72 (app t, J_(2′,1′)=6.4, J_(2′,3′)=4.4, 1H, H2′), 4.65 (m, 1H, H3′),4.33 (m, 1H, H4′), 3.80 (s, 3H, Tr-OMe), 3.20 (s, 3H, OMe), 3.03 (br s,1H, NH), 2.26 (d, J_(5′,5″)=11.7, 1H, H5′), 2.15 (dd, J_(5″,4′)=4.3,J_(5″,5′)=11.7, 1H, H5″), 1.12 (s, 9H, t-Bu).

5′-N-(4-Methoxytrityl)amino-5′-deoxy-N⁶-benzoyl-2′-O-methyladenosine(19). (FIG. 8C) To the solution of 18 (2.7 g, 3 mmol) in THF (30 mL) 1 Mtetrabutylammonium fluoride (TBAF) in THF (6 mL) was added and themixture was stirred at rt 2 h. It was then concentrated to a syrup invacuo. After standard work up and column chromatography using 10-30%gradient THF in CH₂Cl₂ 19 was obtained (1.6 g, 81%) as a colorless foam,¹H NMR δ 8.90 (br s, 1H, NH), 8.14 (s, 1H, H2), 7.98 (s, 1H, H8),8.02-6.79 (m, 19H, aromatic), 5.95 (d, J_(1′,2′)=5.5, 1H, H1′), 4.91(app t, J_(2′,1′)=5.5, 1H, J_(2′,3′)=5.2, H2′), 4.72 (m, 1H, H3′), 4.29(m, 1H, H4′), 3.77 (s, 3H, Tr-OMe), 3.52 (s, 3H, OMe), 3.09 (br s, 1H,NH), 2.67 (d, J_(OH,3)=3.4, 1H, OH3′), 2.60 (dd, J_(5′,5″)=11.7, 1H,H5′), 2.15 (dd, J_(5″,4′)=4.3, J_(5″,5′)=11.7, 1H, H5″), 1.12 (s, 9H,t-Bu).

5′-N-(4-Methoxytrityl)amino-5′-deoxy-N⁶-benzoyl-2′-O-methyladenosine-3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)(20). (FIG. 8C) Using the same procedure as for the preparation of 5, 19(1 g, 1.5 mmol) was converted into 20 and after column chromatographyusing CH₂Cl₂ containing 1% Et₃N (v/v) a colorless foam (0.55 g, 74%) wasobtained, ³¹P NMR δ 151.2 (s), 151.8 (s).

Referring to FIG. 8D, 5′-Deoxy-5′-iodo-2′-O-methyluridine (21). Thiscompound was prepared from 1 using the procedure of Verheyden andMoffatt (J. Org. Chem., 1970, 35, 2319, and is incorporated by referenceherin in its entirety) for selective iodination of thymidine andisolated in 59% yield by column chromatography using 1-5% MeOH in CH₂Cl₂for elution, ¹H NMR (DMSO-d₆) δ 7.76 (d, J_(6,5)=8.1, 1H, H6), 5.94 (d,J_(1′,2′)=5.4, 1H, H1′), 5.77 (d, J_(5,6)=8.1, 1H, H5), 5.52 (d,J_(OH,3′)=6.0, 1H, 3′OH), 4.11 (dd, J_(3′,2′)=5.36, J_(3′,4′)=10.2, 1H,H3′), 4.06 (app t, J_(2′,1′)=5.4, 1H, H2′), 3.93 (m,1H, H4′), 3.63 (dd,J_(5′,4′)=5.4, J_(5′,5″)=10.6, 1H, H5′), 3.49 (dd, J_(5″,4′)=6.9,J_(5″,5′)=10.6, 1H, H5″), 3.42 (s, 3H, OMe).

5′-(S-Triphenylmethyl)mercapto-5′-deoxy-2′-O-methyluridine (22). (FIG.8D) Sodium hydride (52 mg, 2.18 mmol) was suspended in dry DMF (1 mL)under argon at 0° C., and a solution of triphenylmethyl mercaptan (606mg, 2.19 mmol) in dry DMF (7 mL) was added. The mixture was stirred for10 min at rt, cooled in ice and a solution of 21 (690 mg, 1.80 mmol) indry DMF (5 mL) was added. After 3 h at room temperature (rt) solvent wasremoved in vacuo, the residue dissolved in CH₂Cl₂ and washed with 5%aqueous Na₂S₂O₃ and water. The organic layer was dried (Na₂SO₄),evaporated to dryness and chromatographed using 1-2% MeOH in CH₂Cl₂ forelution to afford 22 (860 mg, 68%), mp 187-188° C. (EtOH-H₂O), ¹H NMR δ8.43 (br s, 1H, NH), 7.51-7.29 (m, 16H, Tr, H6), 5.87 (d, J_(1′,2′)=2.4,1H, H1′), 5.78 (d, J_(5,6)=8.1, 1H, H5), 3.90 (m, 1H, H2′), 3.83 (m, 1H,H3′), 3.75 (dd, J_(4′,5′)=2.4, J_(4′,3′)=5.5, 1H, H4′), 2.81 (dd,J_(5′,4=2.4), J_(5′,5″)=13.0, 1H, H5′), 2.52 (dd, J_(5″,4′)=6.6,J_(5″,5′)=13.0, 1H, H5″).

5-(S-Triphenylmethyl)mercapto-5′-deoxy-2′-O-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)(23) (FIG. 8D) Using the same procedure as for the preparation of 5,3′-phosphoramidite 23 was obtained as a white foam in 88% yield afterflash chromatography purification using 50-75% gradient of EtOAc inhexane (1% Et₃N), ³¹P NMR δ 149.1 (s) and 148.7 (s).

Ribozyme Synthesis and Purification

Incorporation of 5′-phosphoramidate at the 5′-end of ribozymes.Synthesis was performed as described (Wincott et al., supra,incorporated by reference herin in its entirety) with a 300 s couplingtime for the 5′-amino phosphoramidites 5, 11 and 20 (FIGS. 8B & 8C).Detritylation was effected using a cycle that consisted of four 10 spulses of TCA, each separated by 7 s wait steps, followed by 30 s ofacetonitrile. This series was then repeated. Finally, the incomingphosphoramidite was coupled for 300 s to complete the synthesis. Theribozyme was base deprotected under standard conditions, however,desilylation was accomplished with TBAF in 24 h rather than HF/TEAsolution.

Incorporation of 5′-amino group at the 5′-end. The synthesis cycle wasmodified slightly from the usual protocol. The 5′-amino phosphoramidites5, 11 and 20 (FIGS. 8B & 8C) were coupled for 300 s. The usual cappingreagent, acetic anhydride, was replaced with t-butylphenoxyaceticanhydride. All ribozymes were synthesized trityl-on. The terminal MMTrgroup was removed upon addition of four 10 s pulses of TCA, eachseparated by 7 s wait steps, followed by 30 s of acetonitrile. Thisseries was repeated until no orange color was observed. The ribozyme wasthen deprotected under standard conditions. In the synthesisincorporating 5, a total of 323 AU of crude material resulted with 41.8%full length product (135 AU). The ribozyme was purifed by anion exchangeHPLC to provide 48 AU of purified ribozyme. Similar recoveries wereobtained with monomers 11 and 20.

Incorporation of bridging 5′-phosphorothioate at the 5%-end. Theoligomers were synthesized using the 5′-thiol phosphoramidite 23 (FIG.8D), coupled for 300 s, and the following amidite coupled for 400 s.Additionally, following the addition of the 5′-thiol amidite, cappingand oxidation, the column was removed from the synthesizer. The cap andfrit were removed, the support was washed out of the column and into anempty syringe with 10 mL of 200 mM AgNO₃ in 1:1 CH₃CN:H₂O. The syringewas capped, wrapped in foil and placed on a shaker for 1 h at rt. Themixture was then replaced into the column. The liquid was removed andthe support was rinsed with 20 mL of 1:1 CH₃CN:H₂O. The support was thentreated with 10 mL 50 mM DTT for 10 min at rt. The support is thenwashed with 20 mL H₂O, then 20 mL CH₃CN. The column was placed on thesynthesizer, washed with CH₃CN for 30 s then reverse flushed for 15 s,this procedure was repeated 4 times. The synthesis was then resumed,with the next phosphoramidite coupling for 400 s and the remainingphosphoramidites coupling for the standard times.

The ribozymes were deprotected with 40% aqueous methylamine for 10 minat 65° C. The silyl groups were removed with TEA/HF solution in 30 minat 65° C. and the oligonucleotides were precipitated from the solution.RPI.4705.5905 yielded 101.5 AU of crude material (half was lost duringdetritylation of 5′-STr) with 16.5% full length product.

Results:

Synthesis of Monomer Building Blocks

The key intermediates for the synthesis of ribozymes containing bridging5′-phosphoramidate and 5′-phosphorothioate linkages were3′-O-phosphoramidites 5, 11, 20 and 23 synthesized according to FIG. 8.

5′-N-(4-Methoxytrityl)amino-5′-deoxy-2′-O-methyluridine Monomer (5)

Uridine derivative 5 was synthesized in a way similar to that reportedby Mag and Engels, supra, for the synthesis of a thymidine analog.5′-Azido derivative 2 (FIG. 8B) was synthesized in one step from2′-O-methyluridine (1) using the procedure of Yamamoto et al., supra.Ammonium hydroxide had to be used instead of water for the hydrolysis ofintermediate 5′-phosphinimide during the conversion of 2 to 3 ((FIG.8B)). It is well documented (Mungall et al., J. Org. Chem. 1975, 40,1659-1662) that nucleoside phosphinimines are relatively stable in watercompared to simple alkyl azides. Protection of the 5′-NH₂ group of 3with 4-methoxytrityl group, followed by standard phosphitylationafforded 3′-O-phosphoramidite 5 in good yield.

5′-N-(4-Methoxytrityl)amino-5′-deoxy-N²-isobutyryl-2′-O-methylguanosineMonomer (11)

Because the one-step procedure for the preparation of the 5′-azidedescribed above does not work well for purine 2′-deoxynucleosides (Maget al., supra), we used a two-step procedure for the introduction of theazido group into the 5′-position of N²-isobutyryl-2′-O-methylguanosine(6) (FIG. 8B). Selective 5′-O-p-toluenesulfonation of 6 at 0° C.afforded the desired mono-substituted derivative 7 in 47% yield and3′,5′-bis-substituted derivative in 15% yield. Attempts to improve theyield and selectivity of this reaction by the portionwise addition ofp-toluenesulfonyl chloride did not help. Displacement of the OTs groupof 7 with an N₃ group using LiN₃ in DMSO proceeded smoothly to yield 8in 78% yield. As in the case of uridine derivative 2 attempts to usetriphenylphosphine in water/pyridine for reduction of 8 to 9 and thusavoid the simultaneous cleavage of the base labile N²-isobutyryl groupfailed to hydrolyze the intermediate 5′-phosphinimine. Thus, catalytichydrogenation of 8 using 10% Pd—C was utilized for the successfulpreparation of 5′-amino-5′-deoxy-2′-O-methyl derivative 9 (80% yield).It is worth noting that 9 underwent a gradual loss of the N²-isobutyrylgroup when left in unbuffered aqueous solution for 16 h or longer. Weattributed this unexpected deacylation to intramolecular base catalysisby the 5′-amino group of 9. Protection of the free amino group of 9 witha 4-methoxytrityl group, followed by phosphitylation afforded3′-O-phosphoramidite 11 in a good yield.

5-N-(4-Methoxytrityl)amino-5′-deoxy-N⁶-benzoyl-2′-O-methyladenosineMonomer (20)

The low selectivity in the tosylation of guanosine derivative 6 promptedus to to use 3′-hydroxyl protection in the preparation of adenosineanalog. Thus, 5′-O-DMT derivative 12 was converted to 3′-O-TBDPSiderivative which was 5′-deprotected to yield 13 with TFA in CH₂Cl₂. Thereaction of 13 with a more reactive sulfonylating agent,p-nitrobenzenesulfonyl chloride, yielded unexpectedly a 2:1 mixture of5′-O-p-nitrobenzenesulfonyl and 5′-chloro-5′-deoxy substitutedderivatives 14 and 15. The mixture was treated with LiN₃ at 80° C.overnight to afford 5′-azido-5′-deoxy derivative 16 in good yield.Catalytic hydrogenation of 16 proceeded smoothly to afford 5′-aminoderivative 17 which was, without purification converted to 5′-N-MMTrprotected derivative 18. Cleavage of the 3′-O-TBDPSi group was achievedusing tetrabutylammonium fluoride and the resulting 19 wasphosphitylated under standard conditions to give the3′-O-phosphoramidite 20 in 74% yield (FIG. 8C).

5′-deoxy-5′-mercapto-2′-O-methyluridine Monomer (23)

Synthesis of the 5′-deoxy-5′-mercapto-2′-O-methyluridine monomer 23started with selective iodination of 2′-O-methyluridine (1) usingmethyltriphenoxyphosphonium iodide as described (Verheyden and Moffat,J. Org. Chem. 1970, 35, 2319-2326 and is incorporated by reference herinin its entirety). The iodo compound 21 was converted in 68% yield intothe 5′-(S-triphenylmethyl)mercapto compound 22 using the sodium salt oftriphenylmethyl mercaptan in DMF as described by Sproat et al., (NucleicAcids Res. 1987, 15, 4837-4848 and is incorporated by reference herin inits entirety). Introduction of an aqueous Na₂S₂O₃ wash into the work upstep was beneficial in reducing the cleavage of STr group and formationof intermolecular disulfide bonds by any iodine present in the reactionmixture (Kamber, Helv. Chim. Acta 1971, 54, 398-422) Phosphitylation of22 under standard conditions (Atkinson, T., Smith, M. In OligonucleotideSynthesis: A Practical Approach, Gait, M. J., Ed.; IRL Press: Oxford,1984, pp 35-81, and is incorporated by reference herin in its entirety)yielded 3′-O-phosphoramidite 23 (FIG. 8D).

Oligonucleotide Synthesis

Synthesis of Oligomers with Bridging 5′-Phosphoramidate

There are four issues that must be addressed when synthesizing oligomerscontaining bridging 5′-phosphoramidate linkages:

1. Coupling of the 5′-amine containing phosphoramidite to the growingchain; 2. Coupling of the following amidite to the 5′-amine; 3.Deprotection conditions; 4. Removal of the MMT protecting group from the5′-amine.

After an extensive study on incorporation of 5′-amino modified monomersinto ribozymes (see Table VI), we found that a coupling time of 300 sfor 5 and 300 s for the following 2′-O-Me nucleotide provided the bestresults. For optimal results, the oligomer was desilylated with TBAFrather then HF/TEA solution as more full length polymer was producedwith the former reagent.

We devised an experiment to study the influence of extended exposure ofthe modified oligonucleotides to the detritylation solution (TCA/CH₂Cl₂)and activator (tetrazole). Following completion of the synthesis, weexposed one oligomer to four “dummy cycles” of detritylation solutionand another to four “dummy cycles” of activator. Although no impact uponfull length product was observed with the extended detritylationexposure, there did appear to be a detrimental effect to extendedexposure to activator.

Finally we investigated the removal of the MMT protecting group. Theoptimal procedure for removal of the MMT group required a “flow through”process. Therefore, detritylation was effected using four 10 s pulses ofTCA with 7 s wait steps between each pulse. This was followed by 30 s ofacetonitrile and then the four 10 s pulses of TCA were repeated. Theincoming amidite was then coupled for 300 s to complete the synthesis.

Synthesis of Oligomers with 5′-amino Group at the 5′-end:

In the process of synthesizing ribozymes containing phosphoramidatelinkages at the 5′-end, we also synthesized ribozymes that contained5′-amines at the 5′-terminus of the ribozyme. The standard syntheticprotocols were modified slightly to optimize synthesis. To ensurecomplete removal of the more stable MMTr protecting group on the5′-amine, the final detritylation step was adjusted as in the previousexample. In addition, t-butylphenoxyacetic anhydride was used as thecapping reagent. We had observed the formation of a side product,identified by MALDI-TOF MS as the N-acetylated ribozyme, when aceticanhydride was the capping agent.

Synthesis of Oligomers with Bridging 5′-phosphorothioates:

A single bridging 5′-phosphorothioate linkage was incorporated into the5′-end of two ribozymes. The 5′-thiol phosphoramidite 23 was coupled for300 s and the following phosphoramidite coupled for 400 s. The ribozymeswere base deprotected as usual and then treated with TEA/HF at 65° C.for 0.5 h rather than 1.5 h. Using the latter reagent we have notobserved substantial cleavage of the P—S bond as observed when TBAF wasused (Sund et al., supra).

Ribozymes containing 5′-amine at the 5′-end showed resistance todigestion by calf spleen 5′-exonuclease equivalent to that observed withP═S backbone modifications. Also, their catalytic activity wascomparable to the wild type ribozymes as described infra.

Example 3 Nuclease Stability, In Vitro Activity and Cell CultureEfficacy of 5′-amino-modified Ribozymes

Materials and Methods:

Radio-labeling of Ribozymes and Substrates. Ribozymes and substrateswere 5′-end-labeled using T4 Polynucleotide Kinase and γ-³²P-ATP. Forinternal labeling, ribozymes were synthesized in two halves with thejunction 5′ to the GAM sequence in Loop II (FIG. 6). The3′-half-ribozyme portion was 5′-end-labeled using T4 PolynucleotideKinase and γ-³²P-ATP, and was then ligated to the 5′-half-ribozymeportion using T4 RNA ligase. Labeled ribozymes were isolated fromhalf-ribozymes and unincorporated label by gel electrophoresis.

Ribozyme Activity Assay. Ribozymes and 5′-³²P-end-labeled substrate wereheated separately in reaction buffer (50 mM Tris-HCl, pH 7.5; 10 mMMgCl₂) to 95° C. for 2 min, quenched on ice, and equilibrated to thefinal reaction temperature (37° C.) prior to starting the reactions.Reactions were carried out in enzyme excess, and were started by mixing˜1 nM substrate with the indicated amounts of ribozyme (50 nM-1 μM) to afinal volume of 50 μL. Aliquots of 5 μL were removed at 1, 5, 15, 30, 60and 120 min, quenched in formamide loading buffer, and loaded onto 15%polyacrylamide/8 M Urea gels. The fraction of substrate and productpresent at each time point was determined by quantitation of scannedimages from a Molecular Dynamics PhosphorImager. Ribozyme cleavage rateswere calculated from plots of the fraction of substrate remaining Vstime using a double exponential curve fit (Kaleidagraph, SynergySoftware). The fast portion of the curve was generally 60-90% of thetotal reaction, so that observed cleavage rates (k_(obs)) were takenfrom fits of the first exponential.

Enzymes. Calf Spleen 5′-exonuclease was purchased from BoehringerMannheim. T4 polynucleotide kinase and Lambda 5′-exonuclease werepurchased from GIBCO/BRL. Enzyme reactions were performed according tothe manufacturers' suggestions.

Cell Culture. Rat aortic smooth muscle cells (SMC) were isolated fromaortic tissue explants from 69-84 day-old female Sprague-Dawley rats(Harlan Sprague Dawley, Inc.) and assayed through passage six. SMC weregrown in Dulbecco's modified Eagle's Medium (DMEM) supplemented withnonessential amino acids (0.1 mM of each amino acid), 0.1 mM sodiumpyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine,20 mM HEPES (all from BioWhittaker) and 10% fetal bovine serum (FBS;Hyclone Laboratories, Inc.).

Preparation of Smooth Muscle Cell Extracts. Rat smooth muscle cellnuclear or total cell extracts were prepared by harvesting SMC from 3confluent T150 flasks. For nuclear lysates, SMC were trypsinized fromthe flasks, washed twice with PBS, and resuspended in 500 μL ofhypotonic buffer. After 40 strokes with a Dounce B homogenizer, 300 μLof 34% sucrose was added and nuclei were pelleted by centrifugation at4° C. and 500×g for 10 min. The nuclei were washed with a solutioncontaining 500 μL of hypotonic buffer and 300 μL of 34% sucrose, thenrepelleted. The pellet was resuspended in buffer A (10 mM Tris-HCl, pH7.5; 400 mM NaCl; 1.5 mM MgCl₂, 0.1 mM EGTA, 5% glycerol, 0.5 mM DTT,and 0.5 mM PMSF) and given 20 strokes in the Dounce B homogenizer. Theresultant suspension was gently shaken for 30 min at 4° C. and thendialyzed at 4° C. for 4 h against 100 mL of dialysis buffer (20 mMTris-HCl, pH 7.5; 0.1 mM EDTA, 75 mM NaCl, 20% glycerol, 0.5 mM DTT and0.5 mM PMSF). After dialysis, the solution was centrifuged at 4° C. and16000×g for 30 min. Aliquots of the supernatant were frozen on dry iceand stored at −70° C. Separate aliquots were used for each assay.

Total cell lysates were prepared by rinsing trypsinized cellpreparations 3×in PBS and pelleting by centrifugation. The pellets wereresuspended in 1 mL of DMEM, 0.5 mM PMSF. PMSF was added as a precautionto minimize proteolytic activity during isolation. Cells werefreeze-thawed 3 times and disrupted by 40 strokes in a Dounce Bhomogenizer. Aliquots of whole cell lysates were aliquoted and frozen at−70° C. Separate aliquots were used for each assay.

Ribozyme Stability Assay. One half pmol of gel-purified, internallylabeled ribozyme was added to 20 μL of reaction buffer (67 mMglycine-KOH [pH 9.4], 2.5 mM MgCl₂, and 50 μg/mL BSA; containing either1 μL of calf spleen 5′-exonuclease [2U/2 mg/mL] or 10 μL of smoothmuscle cell lysate). Samples were placed at 37° C. and 3 μL aliquotswere withdrawn at 0, 30, 60, 120 and 240 min, and 24 h. Aliquots werequenched by the addition of 12 μL of 95% formamide, 0.5×TBE (50 mM Tris,50 mM Borate, 1 mM EDTA) and were frozen prior to gel loading. Ribozymeintegrity was assessed using electrophoresis in 12% acrylamide/7M ureagels. Undigested ribozyme samples were used as size controls. Gels wereimaged by autoradiography.

Proliferation Assays. Cells were plated in growth medium in 24-wellplates at 5×10³ cells per well. After 24 hours, the medium was removed,cells were washed twice with PBS containing Ca²⁺/Mg²⁺, and starvationmedium was added. Starvation medium is growth medium in which theconcentration of FBS is reduced to 0.5%. Cells were starved for 68-72hours before ribozyme treatment. Ribozymes were diluted in serum-freeDMEM with additives as above excluding antibiotics. LipofectAMINE(Gibco-BRL) was added to a final concentration of 3.6 μM DOSPA (=7.2μg/mL LipofectAMINE). Lipid/ribozyme mixtures were vortexed, incubatedfor 15 minutes, and then added to cells which had been washed twice withPBS containing Ca²⁺/Mg²⁺. Cells were incubated with the ribozyme/lipidcomplexes at 37° C. for 4 hours before the mixture was aspirated away.Cells were stimulated by the addition of growth medium. Control cellswere treated with lipid only and stimulated with growth mediumcontaining either 10% or 0% FBS. All conditions were run in triplicate.At the time of stimulation, 5′-bromo-2′-deoxyuridine (BrdU, Sigma) wasadded at a final concentration of 10 μM. Cells were incubated for 24 hand then fixed by the addition of cold 100% methanol plus 0.3% hydrogenperoxide for 30 min at 4° C. The following reagents were used at roomtemperature, unless otherwise noted, to stain the BrdU containingnuclei: i) 2 M HCl for 20 minutes; ii) 1% horse serum in PBS overnightat 4° C.; iii) anti-BrdU monoclonal antibody (Becton-Dickinson) diluted1:200 in 1% bovine serum albumin and 0.5% Tween 20 for 1 hour; iv)biotinylated horse anti-mouse IgG in DPBS for 30 minutes; v) ABC Reagent(Pierce mouse IgG kit) in DPBS for 40 minutes; vi) DAB substrate(Pierce) diluted 1:10 in DAB buffer (Pierce) for 7-10 minutes; and vii)hemotoxylin (Fisher) diluted 1:1 in deionized water for 1-2 minutes. Aminimum of 500 cells per well were counted under the microscope and thepercentage of proliferating cells (BrdU-stained nuclei/total nuclei) wasdetermined.

Resistance of 5′-amino-modified Ribozymes to Digestion by Calf Spleen5′-exonuclease.

Internally-labeled ribozymes were prepared by the separate synthesis of5′- and 3′-half ribozymes, ³²P end-labelling of the 3′-half ribozyme atthe 5′-terminus and subsequent ligation of appropriate 5′- and 3′-halfribozymes to produce a full-length ribozyme with an internal ³²P label.For stabilization against digestion by 3′-exonuclease, the 3′-ends ofall ribozymes were capped with a 3′-3′ linked abasic residue (FIG. 11B;Beigelman et al., 1995 supra). Unless otherwise noted, nonessentialresidues contained 2′-O-Me modifications, while essential residuescontained 2′-ribose moieties as illustrated in FIG. 6. Modifications toribozymes at positions 2.1-2.7 and substitutions at positions U4 and U7are summarized in Table II. While ribozymes containing either ribose(Rz 1) or deoxyribose (Rz 2) moieties at positions 2.1-2.7 were rapidlydigested by calf spleen 5′-exonuclease, ribose containing ribozymesappeared to be more resistant to digestion. 2′-O-Me modification atpositions 2.1-2.7 (Rz 3) slowed digestion but did not preventnucleolytic loss of the Stem I region after extended incubation withcalf spleen exonuclease. Analysis of the digestion patterns revealedthat progressive exonucleolytic digestion within each of these ribozymesstopped near the U4-amino modified residues. Identification of the U4position as the limiting site for exonuclease digestion was achieved bycounting down the digestion ladders of Rzs 1 and 2 on a gel.

Ribozymes containing partial P═S backbone (positions 2.1-2.7, Rz 4) or5′-amino (Rz 6) modifications were resistant to digestion by exonucleaseeven after a 24 h incubation with the calf spleen enzyme. Although thedata discussed used ribozymes containing U4/U7 amino substitutions, wefound that U4-C-allyl modified ribozymes with similar P═S or 5′-aminomodifications were also stable to 5′-exonucleolytic attack (e.g., Rz 8).A low level of contaminating endonuclease activity was observed in theseassays and accounts for the decreased amounts of full-length P═S or5′-amino modified ribozymes after 24 h of incubation. Similar patternsof nuclease resistance were observed for these ribozymes in parallelassays using Lambda 5′-exonuclease.

Ribozyme Stability in Rat Smooth Muscle Cell Lysates.

Internally-labeled ribozymes were prepared for lysate stability assaysas described in the previous section and in Materials and Methods. The3′-ends of all ribozymes contained a 3′-3′ linked abasic residue. Riboseand 2′-O-Me substitutions into the ribozyme used standard patterns whichwere discussed above. Modifications to positions 2.1-2.7 and 5′-endsubstitution for the ribozymes are summarized in Table II. The data showthat ribozymes containing unprotected ribose (Rz 1) or deoxyribose (Rz2) residues in positions 2.1-2.7 are digested in both nuclear and wholecell lysates, but at a much slower rate than was observed in assayscontaining purified calf spleen 5′-exonuclease. Incubation of theseribozymes in SMC lysates resulted in the progressive shortening ofribozyme fragments over time, suggesting that the molecules were beingdigested by a cellular 5′-exonuclease activity. While progressive 3′-enddigestion by an uncharacterized cellular enzyme cannot be ruled out inthese assays, previous results in serum and cell extracts have shownthat the addition of a 3′-3′ abasic residue at the 3′-terminus rendersribozymes resistant to 3′-exonucleolytic attack (Beigelman et al., 1995supra).

Neither 2′-O-Me (Rz 3), P═S backbone (Rz 4) or 5′-amino (Rzs 6 and 8)modification of ribozymes totally protected the molecules from digestionin SMC extracts. An examination of the digestion patterns revealed thatwhile there was no exonucleolytic cleavage of these ribozymes, they werefragmented by endonucleolytic attack. 2′-substitution for theU4/U7-amino groups of Rz 6 using U4/U7-C-allyl/O-Me groups of Rz 8 didnot affect the resistance of 5′-amino containing ribozymes toexonucleolytic attack. Taken together with the data from the previoussection, these data show that while 2′-O-Me modification can providelimited protection against 5′-exonucleolytic digestion in cellularextracts, 2′-O-Me substitution provides much less protection versusdigestion by purified 5′-exonuclease. In contrast, P═S backbone and5′-amino modifications prevented digestion by both purified calf spleen5′-exonuclease and SMC 5′-exonuclease(s) but provided little addedprotection from endonucleolytic attack at the essential ribose residues(positions 5, 6, 8, 12 and 15.1). Based on these data and previousreports of the ability of U4/U7 modifications to restrictendonucleolytic attack at essential ribose residues (Beigelman et al.,1995 supra), we conclude that the effects of P═S and 5′-aminosubstitutions are confined to a very localized region at the 5′-end ofthe ribozyme.

The digestion profiles of ribozymes containing ribose (Rz 1) ordeoxyribose (Rz 2) residues at positions 2.1-2.7 were quite different inthe two SMC lysates. Although there was approximately 10 times moreprotein in the cellular lysates than in the nuclear lysates, this alonecannot account for the differences, because the degree of digestion forRz 2 in cellular lysates was more than 10× greater than greater innuclear lysates. In contrast, the degree of digestion for Rz 1 wasapproximately the same in both lysates at all times. These data suggestthat nucleolytic digestion of ribozymes in SMC lysates is highlydependent upon the chemical nature of the ribozymes. Differences in thedigestion patterns of Rz 1 and Rz 2 suggest that different enzymes maybe responsible for the exonucleolytic digestion near the 5′-regions ofthese molecules. This differential chemical susceptibility of ribozymesto nucleolytic digestion was even more obvious when other cell lysateswere used for comparison and in some cases (e.g., HL60 cell lysates) theribose-containing Stem I regions were more susceptible to digestion thanthe deoxyribose-containing stems. Such comparative data show that thesusceptibility of ribozymes to digestion by cellular nucleases is highlydependent upon both cell type and chemical modification to the ribozyme.

On the basis of the nuclease assays, we conclude that 1) 5′-aminomodified ribozymes are as resistant to 5′-exonucleolytic digestion asthioated ribozymes, and 2) the advantage which P═S modifications give toribozyme efficacy in cells is not just a result of their superiornuclease stability, but probably also results from intracellularlocalization or protein association which is mediated by the thioatemoieties within the ribozymes.

Catalytic Activity of 5′-amino Modified Ribozymes.

The relative effect of 5′-amino substitution on ribozyme catalyticactivity was investigated under standard assay conditions as described,supra, in Materials and Methods. The catalytic activity of each ribozymewas assayed at two concentrations and the results were plotted todetermine the region of the reaction which gave exponential rates ateach concentration. Cleavage rates (k_(obs) values) were calculated fromfits of the first exponential. Table III shows an activity comparisonfor the five U4/U7-amino containing ribozymes at concentrations of 40and 500 nM (roughly 4 and 50 fold above K_(M)). Activity is presentedboth as the cleavage rate (min⁻¹) and as a percentage of the rate forthe control, Rz 3.

Comparison of the catalytic rates of selected ribozymes from Table IIrevealed that neither P═S nor 5′-amino modification of Rz 3 (Rzs 4 and6, respectively) affected the catalytic rate significantly. Ribozymescontaining 2′-O-Me substitutions at positions 2.1-2.7 (Rz 3) (FIG. 6)showed slightly better catalytic activity (20-30%) in this assay thanribozymes containing ribose moieties at these positions (Rz 1). Asreported earlier (Beigelman et al., 1995 supra), we generally see verysimilar catalytic rates for ribozymes containing ribose and 2′-O-Mesubstitutions at positions 2.1-2.7 (FIG. 6) although there are generallyalso substitutions at positions 15.1-15.7 (FIG. 6) in the moleculeswhich have been compared. The k_(obs) values for P═S and 5′-aminomodified ribozymes (Rz 4 and 6, respectively) were equivalent, withinerror, to those of the ribose-containing Rz 1.

The deoxyribose-substituted Rz 2 is peculiar in that it showed a 6-10fold reduction in activity when compared with the other 2.1-2.7 position(FIG. 6) substitutions (Rzs 1, 3, 4, and 6). The similarity in cleavagerates at 40 and 500 nM for this ribozyme suggest that the reducedk_(obs) for Rz 2 was not a result of reduced binding affinity but morelikely reflects a 6-10 fold decrease in k_(cat).

This data represents the first comparative report of the effects ofsubstitution at positions 2.1-2.7 into ribozymes using U4/U7-amino (orU4/N₇-amino) stabilized ribozymes and additionally demonstrates thatnuclease stabilizing modifications can be used to replace P═S backbonesubstitutions in ribozymes without reducing catalytic activity.

Cellular Efficacy of 5′-amino Modified Ribozymes.

Based on catalytic data (Table III) and the increased stability observedwith 5′-amino modified Rz 6 and 8 in the nuclease assays, we decided tocompare the efficacy of Rz 6 to the thioated Rz 4 in cell assays ofribozyme activity. The relative abilities of ribozymes containingvarious modifications at positions 2.1-2.7 (FIG. 6) and/or the5′-terminus were compared in a cell proliferation assay using rat smoothmuscle cells. Ribozymes were delivered using lipofectAMINE as described,supra, in the Materials and Methods section. After the application ofribozymes, cells were metabolically labeled with BrdU for 24 h and thenumber of proliferating SMC nuclei were determined by differentialstaining using an anti-BrdU antibody detection system and hematoxylin.

Ribozymes containing ribose (Rz 1), deoxyribose (Rz 2) or 2′-O-Memodified (Rz 3) nucleosides at positions 2.1-2.7 as well ascatalytically inactive (Rzs 5, 7, 9, and 11) were included as controlsfor non-specific ribozyme inhibition. The stability data suggested thatRz 1 and 2 would be unstable in SMC, and previous results comparingthioated and nonthioated ribozymes suggested that even though Rz 3 isrelatively nuclease-resistant in the SMC lysates, nonthioated ribozymeswould be less effective in cellular assays. Ribozymes with catalyticallyinactivated core regions (Rz 5, 7, 9, and 11) were included todifferentiate true ribozyme activity from non-specific phosphorothioateeffects. Ribozymes with catalytically active cores containing eitherU4/U7-amino or U4/U7-C-allyl-O-Me modifications and P═S (Rz 4 and Rz 10,respectively) or 5′-amino modifications (Rz 6 and Rz 8, respectively)were included as positive controls. The relative abilities of eachribozyme to inhibit SMC proliferation are summarized in Table IV andshown graphically in FIGS. 9 and 10.

As shown in Table IV, ribozymes with ribose (Rz 1), deoxyribose (Rz 2)or 2′-O-Me (Rz 3) moieties at positions 2.1-2.7 exhibited similarly lowlevels of inhibitory activity in the SMC proliferation assay. Thedeficiency of inhibitory action by either Rz 1 or Rz 2 reflected theinherent nuclease susceptibility of these molecules in SMC lysates andsuggested that even the low levels of nuclease activity which weobserved in the lysates may be enough to digest unstablized ribozymesquickly within the cellular enivironment. Alternatively, Rzs 1 and 2 maybe showing lower efficiency of inhibition of cellular proliferationbecause they are not localized near target molecules. The lower efficacywith Rz 3 is consistent with this latter hypothesis. Based upon our datashowing the resistance of Rz 3 to digestion using purified preparationsof calf spleen 5′-exonuclease, these molecules are expected to berelatively stable within the cells, yet they don't decrease cellularproliferative rates any better than Rzs 1 or 2. We feel that Rz 3preparations are stable within cells and the decreased inhibitoryactivity may be because of issues unrelated to their nucleasesusceptibility.

Comparison of the relative efficacies showed that U4/U7-amino containing5′-amino-modified Rz 6 was as effective at inhibiting SMC proliferationas the thioate-stabilized Rz 4. Both of these molecules were moreeffective than the 5′-amino, U4-C-allyl modified Rz 8, which wasslightly more active than Rzs 1-3. Further, Rz 6, but not Rz 4 showedbetter efficacy than their catalytically inactive counterparts, Rzs 7and 5, respectively. These data show that P═S modifications of ribozymesenhance their cellular efficacy over that seen with non-stabilizedribozymes. Similar efficacies can be achieved without the apparentnon-specific effects of the thioated compounds when othernuclease-stabilizing chemistries are present within the ribozymestructure (e.g., the 5′-amino modification). The further observationthat nuclease-stable, Rz 5 exhibited better inhibitory activity thannuclease-sensitive, catalytically active Rzs 1 and 2 shows that nucleasestabilization is important for efficient ribozyme efficacy in cells whenthe ribozymes are delivered exogenously.

In summary, we have found that 5′-amino, U4/U7-amino modified ribozymesexhibited in vitro stability, in vitro catalytic activity and cellularefficacy (FIGS. 9 and 10) which was equivalent to similar thioated,U4/U7-amino modified ribozymes. Additionally, 5′-amino containingribozymes showed slightly better cellular efficacy when using theU4/U7-amino format (FIG. 9, 5′-amino Active RZ) than with the U4-C-allylformat (FIG. 10, 5′-amino Active RZ). This latter observation reflectedslightly better in vitro catalytic activities which were observed withthe U4/U7-amino compounds.

Taken together, these data support the notion that a 5′-aminomodification to ribozymes will enhance their intracellular stability andenable intracellular efficacy in a manner which is consistent with theirobserved relative catalytic rates in vitro. Although it is not possibleto determine on the basis of these studies whether 5′-amino containingribozymes colocalize to the same intracellular region as thioatedribozymes, these results do suggest that 5′-amino modified ribozymes canbe used effectively in animal studies of ribozyme efficacy withoutexhibiting some of the concentration dependent non-specific effectswhich have been observed by others when using thioated antisenseoligonucleotides.

Example 4 Terminal Modification of Ribozymes Using Phosphorothioates

Comparison of 5′-end versus 3′-end modifications—Ribozymes targetingc-myb site 575, as described in Example in 3, supra, were complexed withLipofectAMINE and delivered to rat aortic smooth muscle cells at a 100nM dose. Cell proliferation was measured as described in Materials andMethods of Example 3, supra. Active and inactive versions of severaldifferent chemical modifications were tested. “2′-O-Me” indicates an RNAcore with five 2′-O-methyl residues at the 5′- and 3′-ends. “2′-O-MeP═S” indicates an RNA core with five 2′-O-methyl phosphorothioateresidues at the 5′- and 3′-ends. “U4 C-allyl” and “U4 C-allyl P═S”indicate U4 and U7 2′-C-allyl “stabilized” cores without and withphosphorothioate linkages at the 5′- and 3′-ends, respectively. “U4,7NH₂” and “U4,7 NH₂ P═S” indicate U4 and U7 2′-amino “stabilized” coreswithout and with phosphorothioate linkages at the 5′- and 3′-ends,respectively. Relative smooth muscle cell proliferation is calculated asfollows: (% proliferation with ribozyme−% basal proliferation)÷(%proliferation with serum−% basal proliferation)×100.

The results indicate that both a nuclease-resistant core andphosphorothioate linkages in the binding arms are necessary forsignificant cell culture efficacy when the ribozymes are deliveredexogenously. Since phosphorothioate linkages may be associated with somedegree of cytotoxicity and some non-specific effects [Uhlmann et al.,1990 Chem. Rev. 90, 543], we wished to determine the minimum number ofphosphorothioates sufficient for ribozyme-mediated cell efficacy. Acomparison of ribozymes containing either 5 phosphorothioate linkages atthe 5′-end, or 5 phosphorothioate linkages at the 3′-end, or 5phosphorothioate linkages at both the 5′- and 3′-ends. The ribozymecontaining phosphorothioates only at the 3′-end showed only marginalefficacy when compared with an inactive ribozyme, while the ribozymecontaining phosphorothioates at the 5′-end showed equivalent efficacy tothat containing phosphorothioates at both the 5′- and 3′-ends. In thisexperiment, the inactive ribozyme showed some inhibition relative to thevehicle-treated control. A ribozyme with scrambled sequence binding armsexhibited an equivalent degree of inhibition to an inactive ribozyme,indicating that this effect was not mediated by ribozyme binding, butwas truly a “non-specific” effect on proliferation. Next, we comparedribozymes with varying numbers of phosphorothioates at the 5′-end. Thedegree of efficacy gradually decreased as the number of phosphorothioatelinkages was reduced. From these experiments we concluded that a minimumof four to five phosphorothioate linkages at the 5′-end is sufficient tomaintain optimal efficacy.

The ribozymes used in this study contained either 3′-phosphorothioatelinkages, or a 3′-3′ “inverted thymidine” modification to protectagainst 3′-exonuclease activity. We have subsequently shown that theoutcome of this assay is not particularly sensitive to the presence orabsence of this 3′-protecting group. C-myb ribozymes containing variousprotecting groups including a 3′-3′ inverted thymidine, a 3′-3′ invertedabasic residue, a 3′-butanediol showed equivalent efficacy in inhibitingsmooth muscle cell proliferation.

Example 5 Incorporation of Phosphorodithioate Linkages into Ribozymes

Materials and Methods

Referring to FIG. 12, 2′-O-TBDMS-5′-O-DMT-N-protected ribonucleosides,5′-O-DMT-N-protected deoxy- and 2′-O-Me ribonucleosides were from ChemGenes Corporation, Waltham, Mass. Commercially available anhydroussolvents were employed without purification. Concentrations of solutionswere carried out in vacuo at 40° C. or lower using an aspirator or anoil vacuum pump. Solids were dried at room temperature in a desiccatorover phosphorus pentoxide and potassium hydroxide. ³¹P NMR spectra wererecorded on a Varian Gemini 400 spectrometer operating at 161.947 MHzwith 85% phosphoric acid as external standard. Oligonucleotides weresynthesized on an Applied Biosystems 381A synthesizer using AppliedBiosystems columns.

General Procedures

Ribonucleoside 3′-S-(2-cyanoethyl)N,N-dimethylthiophosphoramiditeSynthesis

Suitably protected 2′-t-butyldimethylsilyl-5-O′-dimethoxytritylnucleoside (2.0 mmol) (FIG. 12) was dried and was dissolved in drydichloromethane (CH₂Cl₂) (20 ml) under argon and the solution was cooledto 0° C. (ice-bath). The mixture of N,N-diisopropylethylamine (DIPEA)(0.56 ml, 3.20 mmol) and N,N,N′,N′-tetramethylchlorophosphordiamidite[PCI(NMe₂)₂] (0.40 g, 2.60 mmol) in dry CH₂Cl₂ (5 ml) was added dropwiseto the above solution under constant stirring. The mixture was stirredat rt for 30 min after which time β-mercaptopropionitrile (0.44 g, 5.0mmol) was added and the reaction mixture was stirred at rt foradditional 1 h. The mixture was then poured into CH₂Cl₂ (100 ml, 1%triethylamine) and washed with saturated NaHCO₃ (100 ml), 10% aq. Na₂CO₃(2×100 ml) and saturated brine (100 ml). The organic layer to which 1 mlof Et₃N was added was dried (Na₂SO₄) for 20 min and concentrated to ca10 ml in vacuo. This solution was added dropwise into the stirred,cooled (0° C.), degassed hexanes (200 ml, 1% Et₃N). The precipitate wasfiltered off and dried in vacuo to yield the product as a white powder.

2′-Deoxy and 2′-O-methylribonucleoside3′-S-(2-cyanoethyl)N,N-dimethylthiophosphoramidite Synthesis

Suitably protected 5′-O-dimethoxytrityl nucleoside (4 mmol) and DIPEA(1.05 ml, 6.0 mmol) were dried and were dissolved in dry CH₂Cl₂ (30 ml)under Ar and the solution was cooled to 0° C. (ice-bath). PCI(NMe₂)₂(0.62 g, 4.0 mmol) was added dropwise under stirring. The clear solutionwas stirred at rt for 10 min, then β-mercaptopropionitrile (0.42 g, 4.8mmol) was added and the solution was stirred at rt for additional 1 h.The work up of the reaction mixture as described for ribonucleosidesabove yielded products as white powders.

Synthesis with Manual Thiolation:

Model syntheses of ribo and 2′-O-methyl dithioate oligonucleotidesequences was performed on an ABI model 394 synthesizer using a modifiedsynthesis cycle for thiolation. A 10 μmol cycle was created toaccomodate manual sulfurization off of the instrument. This wasaccomplished by placing an interrupt step immediately after thephosphoramidite coupling step following the final acetonitrile wash andargon flush. The synthesizer column containing the oligo bound solidsupport was subsequently removed from the instrument. One frit was thenremoved from the end of the column and a 20 ml syringe attached to thatend. At the other end of the column (the end with a frit) was attached a20 ml syringe containing a solution of 1.5 g elemental sulfur dissolvedin 20 ml of carbon disulfide and 2,6-lutidine (1:1 by volume). Byforcing the thiolation solution through the column, the support wastransferred to the empty syringe. This syringe, now containing thesupport suspended in thiolation solution, was capped off and placed onan orbital shaker for one hour. The syringe containing the suspendedsupport was then reattached to the end of the column without a frit andthe contents transferred back to the column. A new frit was then placedon the column. Excess sulfur was then washed off the support with a 20ml solution of carbon disulfide/2,6-lutidine 1:1 followed by 20 mlanhydrous acetonitrile. Synthesis was then resumed by placing the columnback on the instrument. The synthesizer cycle was resumed and the entireprocess repeated as necessary for each dithioate substitution introducedinto the oligo. It should be noted that a 300 second coupling time wasutilized for 2′-O-methyl residues while a 600 second coupling time wasutilized for ribo residues. Also, the use of S-ethyl tetrazole wasavoided in order to minimize side reactions resulting from the morelabile dimethylamino substituted phosphoramidite moiety. Also noteoxidation prior to capping in the cycle. Cleavage from the support anddeprotection results from treatment of the solid support with a solutionof 15% benzene or toluene in saturated ethanolic ammonia (−70° C. sat.)for 2 hours at rt and 15 hours at 55° C. Our studies demonstrate 90%thiolation efficiency under these conditions as determined by 31P NMRanalysis of crude material.

Synthesis with Automated Thiolation:

A new synthesizer cycle (2.5 μmol) was created for fully automatedsynthesis of 2′-O-methyl and ribo phophorodithioate oligonucleotides.Tetrazole was used in place of S-ethyl tetrazole to minimize sidereactions. The following bottle positions on the ABI 394 synthesizerwere assigned to the following solutions:

-   -   position #10: carbon disulfide:pyridine:TEA, elemental sulfur        (95:95:10, 5%) Note: this solution must be used within 24 hours        for optimum results.    -   position #15: carbon disulfide    -   position #19: dichloromethane

The synthesis cycle was designed to deliver 12 equivalents or less ofphosphoramidite with 600 second coupling times for ribo residues and 300second coupling times for 2′-O-methyl residues. After coupling,thiolation solution (bottle #10) is delivered in two pulses. In ourstudies, the thiolation time was varied between 1 and 60 minutes, withan optimum time of 6 minutes. Care must be taken to avoid precipitationof sulfur in the synthesizer lines; as such, carbon disulfide (bottle#15) washes precede and follow delivery of the thiolation solution.Dichloromethane washes (bottle #19) are used to remove excess carbondisulfide from the column. In our studies, oxidation with aqueousiodine/pyridine followed standard capping in order to visualizeincomplete thiolation by 31P NMR. This step was necessary foroptimization, but is to be removed from standard synthetic dithioateprotocols due to the increased possibility of phosphorothioate andphosphodiester contamination. Cleavage from the support and deprotectionresults from treatment of the solid support with a solution of 15%benzene or toluene in saturated ethanolic ammonia (−70° C. sat.) for 2hours at rt and 15 hours at 55° C. Our studies demonstrate 90%thiolation efficiency under these conditions as determined by 31P NMRanalysis of crude material.

Example 6 General Procedure for the Synthesis of Carbocyclic NucleosidePhosphoramidites

Referring to FIG. 14, carbocyclic nucleosides (1) are synthesizedessentially as described by Agrofoglio et al., 1994, Tetrahedron 50,10611. Carbocyclic nucleosides (1) were 5′-protected for example by5′-O-dimethoxytritylating 1 according to the standard procedure (seeOligonucleotide Synthesis: A Practical Approach, M. J. Gait Ed.; IRLPress, Oxford, 1984, p 27, and is incorporated by reference herin in itsentirety) to yield 2 in high yield in the form of yellowish foams aftersilica gel column chromatography. To the stirred solution of theprotected nucleoside 2 in 50 mL of dry THF and pyridine (4 eq), AgNO₃(2.4 eq) was added. After 10 minutes tert-butyldimethylsilyl chloride(1.5 eq) was added and the reaction mixture was stirred at roomtemperature for 12 hours. The resulted suspension was filtered into 100mL of 5% aq NaHCO₃. The solution was extracted with dichloromethane(2×100 mL). The combined organic layer was washed with brine, dried overNa₂SO₄ and evaporated. The residue containing 3 was purified by flashchromatography on silica gel. Compound 3 was then phosphitylated in thefollowing way: To the ice-cooled stirred solution of protectednucleoside 3 (1 mmol) in dry dichloromethane (20 mL) under argon blanketwas added dropwise via syringe the premixed solution ofN,N-diisopropylethylamine (2.5 eq) and 2-cyanoethylN′,N-diisopropylchlorophosphoramidite (1.2 eq) in dichloromethane (3mL). Simultaneously via another syringe N-methylimidazole (1 eq) wasadded and stirring was continued for 2 hours at room temperature. Afterthat the reaction mixture was again ice-cooled and quenched with 15 mlof dry methanol. After 5 min stirring, the mixture was concentrated invacuo (<40° C.) and purified by flash chromatography on silica gel togive corresponding phosphoroamidite 4.

Carbocyclic nucleoside phosphoramidites are incorporated into ribozymesusing solid phase synthesis as described by Wincott et al., 1995 supra,incorporated by reference herein in its entirety. The ribozymes aredeprotected using the standard protocol described above.

Example 7 General Procedure for the Synthesis of Alpha NucleosidePhosphoramidites

Referring to FIG. 15, alpha nucleosides (1) are synthesized essentiallyas described by Debart et al., 1992, Nucleic Acid Res. 20, 1193; andDebart et al., 1995, Tetrahedron Lett. 31, 3537. Alpha nucleosides (1)were 5′-protected for example by 5′-O-dimethoxytritylating 1 accordingto the standard procedure (see Oligonucleotide Synthesis: A PracticalApproach, M. J. Gait Ed.; IRL Press, Oxford, 1984, p 27, and isincorporated by reference herin in its entirety) to yield 2 in highyield in the form of yellowish foams after silica gel columnchromatography. To the stirred solution of the protected nucleoside 2 in50 mL of dry THF and pyridine (4 eq), AgNO₃ (2.4 eq) was added. After 10minutes tert-butyldimethylsilyl chloride (1.5 eq) was added and thereaction mixture was stirred at room temperature for 12 hours. Theresulted suspension was filtered into 100 mL of 5% aq NaHCO₃. Thesolution was extracted with dichloromethane (2×100 mL). The combinedorganic layer was washed with brine, dried over Na₂SO₄ and evaporated.The residue containing 3 was purified by flash chromatography on silicagel. Compound 3 was then phosphitylated in the following way: To theice-cooled stirred solution of protected nucleoside 3 (1 mmol) in drydichloromethane (20 mL) under argon blanket was added dropwise viasyringe the premixed solution of N,N-diisopropylethylamine (2.5 eq) and2-cyanoethyl N′,N-diisopropylchlorophosphoramidite (1.2 eq) indichloromethane (3 mL). Simultaneously via another syringeN-methylimidazole (1 eq) was added and stirring was continued for 2hours at room temperature. After that the reaction mixture was againice-cooled and quenched with 15 ml of dry methanol. After 5 minstirring, the mixture was concentrated in vacuo (<40° C.) and purifiedby flash chromatography on silica gel to give correspondingphosphoroamidite 4.

Alpha nucleoside phosphoramidites are incorporated into ribozymes usingsolid phase synthesis as described by Wincott et al., 1995 supra, and isincorporated by reference herin in its entirety. The ribozymes aredeprotected using the standard protocol described above.

Example 8 General Procedure for the Synthesis of1-(β-D-erythrofuranosyl) Nucleoside Phosphoramidites

Referring to FIG. 16, 1-(β-D-erythrofuranosyl) nucleosides (1) aresynthesized essentially as described by Szekeres et al, 1977, J.Carbohydr. Nucleosides Nucleotides. 4, 147. 1-(β-D-erythrofuranosyl)nucleosides (1) were treated with AgNO₃ (2.4 eq). After 10 minutestert-butyldimethylsilyl chloride (1.5 eq) was added and the reactionmixture was stirred at room temperature for 12 hours. The resultedsuspension was filtered into 100 mL of 5% aq NaHCO₃. The solution wasextracted with dichloromethane (2×100 mL). The combined organic layerwas washed with brine, dried over Na₂SO₄ and evaporated. The residuecontaining 2 was purified by flash chromatography on silica gel.Compound 2 was then phosphitylated in the following way: To theice-cooled stirred solution of protected nucleoside 2 (1 mmol) in drydichloromethane (20 mL) under argon blanket was added dropwise viasyringe the premixed solution of N,N-diisopropylethylamine (2.5 eq) and2-cyanoethyl N′,N-diisopropylchlorophosphoramidite (1.2 eq) indichloromethane (3 mL). Simultaneously via another syringeN-methylimidazole (1 eq) was added and stirring was continued for 2hours at room temperature. After that the reaction mixture was againice-cooled and quenched with 15 ml of dry methanol. After 5 minstirring, the mixture was concentrated in vacuo (<40° C.) and purifiedby flash chromatography on silica gel to give correspondingphosphoroamidite 3.

1-(β-D-erythrofuranosyl) nucleoside phosphoramidites are incorporatedinto ribozymes using solid phase synthesis as described by Wincott etal., 1995 supra. The ribozymes aree deprotected using the standardprotocol described above.

Example 9 General Procedure for the Synthesis of Inverted Deoxyabasic5′-O-Succinate and 5′-O-Phosphoramidite

Referring to to FIG. 17, commercially available 2-deoxyribose isconverted to compound 1 in a two step process. In the first step,2-deoxyribose is treated with a mixture of acetyl chloride and methanol.In the second step, the reaction mixture is treated with p-toluoylchloride/pyridine mixture to yield 1. Compund 1 is incubated with amixture of triethyl silane and boron trifluoride in ethanol to yieldcompound 2. Treatment of 4 with sodium methylate in methanol yieldcompound 3. Reacting 3 with t-butyl-diphenyl-silyl chloride in pyridineyields compound 4. The 3′-end of 4 is tritylated using4,4′-dimethoxytrityl chloride in pyridine to yield compound 5. The5′-protecting group in 5 can be removed using a mixture oftriethylamine/hydrogen fluoride/DCM to yield 6.

A succinate group can be attached to the 5′-end of compound 6 byreacting the compound with a mixture of succinic anhydride and4-dimethylaminopyridine to yield compound 7.

Compound 6, can be converted into a phosphoramidite by standardphosphitylation reaction described supra to yield compound 8. Reactionof 8 with a standard phosphoramidite will yield a 5′-5′-inverted abasicdeoxyribose linkage as shown in FIG. 7C.

Example 10 General Procedure for the Synthesis of 3′-2′-InvertedNucleotide or 3′-2′-Inverted Abasic Linkage

Refering to FIG. 13, a commercially available5′-dimethoxytrityl-3′-silyl-containing nucleoside (1) is treated with astandard phosphitylation reagent such as 2-cyanoethylN′,N-diisopropylchlorophosphoramidite to yield compound 2. Reaction ofcompound 3, wherein B is a natural or a modified base (described inSeliger et al., Canadian Patent Application Publication No. 2,106,819.,and is incorporated by reference herein), with compound 2 will result ina 3′-2′-inverted nucleotide linkage as shown in FIG. 11B (3′-2′-invertednucleotide).

Reaction of compound 3, wherein B is H (see FIG. 17; compound 7), withcompound 2 will result in a 3′-2′-inverted abasic linkage as shown inFIG. 11B (3′-2′-inverted abasic).

Refering to FIG. 17, compound 7 can be reacted with compound 2 in FIG.13 to yield a 3′-2′-inverted abasic deoxyribose linkage as shown in FIG.11B.

Alternatively, 7 (FIG. 17) can be reacted with a standard nucleosidephosphoramidite to yield a 3′-3′-inverted abasic deoxyribose linkage asshown in FIG. 11B.

Example 11 In Vitro RNA Cleavage Activity of Ribozymes with 5′-TerminalPhosphorodithioate Modifications

Radio-labeling of Ribozymes and Substrates. Substrates were5′-end-labeled using T4 Polynucleotide Kinase and γ-³²P-ATP.

Ribozyme Activity Assay. Ribozymes and 5′-³²P-end-labeled substrate wereheated separately in reaction buffer (50 mM Tris-HCl, pH 7.5; 10 mMMgCl₂) to 95° C. for 2 min, quenched on ice, and equilibrated to thefinal reaction temperature (37° C.) prior to starting the reactions.Reactions were carried out in enzyme excess, and were started by mixing˜1 nM substrate with the indicated amounts of ribozyme (50 nM-1 μM) to afinal volume of 50 μL. Aliquots of 5 μL were removed at 1, 5, 15, 30, 60and 120 min, quenched in formamide loading buffer, and loaded onto 15%polyacrylamide/8 M Urea gels. The fraction of substrate and productpresent at each time point was determined by quantitation of scannedimages from a Molecular Dynamics PhosphorImager. Ribozyme cleavage rateswere calculated from plots of the fraction of substrate remaining vstime using a double exponential curve fit (Kaleidagraph, SynergySoftware). The fast portion of the curve was generally 60-90% of thetotal reaction, so that observed cleavage rates (k_(obs)) were takenfrom fits of the first exponential.

Referring to FIG. 18, ribozymes with either one or twophosphorodithioate substitutions were capable of catalyzing efficientRNA clevage reactions. The results show that modification of ribozymesat the 5′-end do not significantly effect the activity of ribozymes.

Uses

The 5′- and/or 3′-substituted enzymatic nucleic acids of this inventioncan be used to form stable molecules with enhanced activity as discussedabove for use in enzymatic cleavage of target RNA. Such nucleic acidscan be formed enzymatically using triphosphate forms by standardprocedure. Administration of such nucleic acids into cells is bystandard methods. Their in vitro utility is as known in the art. SeeSullivan et al., PCT WO 94/02595.

Diagnostic Uses

Enzymatic nucleic acids of this invention may be used as diagnostictools to examine genetic drift and mutations within diseased cells or todetect the presence of target RNA in a cell. The close relationshipbetween ribozyme activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple ribozymes described in this invention, one may map nucleotidechanges which are important to RNA structure and function in vitro, aswell as in cells and tissues. Cleavage of target RNAs with ribozymes maybe used to inhibit gene expression and define the role (essentially) ofspecified gene products in the progression of disease. In this manner,other genetic targets may be defined as important mediators of thedisease. These experiments will lead to better treatment of the diseaseprogression by affording the possibility of combinational therapies(e.g., multiple ribozymes targeted to different genes, ribozymes coupledwith known small molecule inhibitors, or intermittent treatment withcombinations of ribozymes and/or other chemical or biologicalmolecules). Other in vitro uses of ribozymes of this invention are wellknown in the art, and include detection of the presence of mRNAsassociated with disease condition. Such RNA is detected by determiningthe presence of a cleavage product after treatment with a ribozyme usingstandard methodology.

In a specific example, ribozymes which can cleave only wild-type ormutant forms of the target RNA are used for the assay. The firstribozyme is used to identify wild-type RNA present in the sample and thesecond ribozyme will be used to identify mutant RNA in the sample. Asreaction controls, synthetic substrates of both wild-type and mutant RNAwill be cleaved by both ribozymes to demonstrate the relative ribozymeefficiencies in the reactions and the absence of cleavage of the“non-targeted” RNA species. The cleavage products from the syntheticsubstrates will also serve to generate size markers for the analysis ofwild-type and mutant RNAs in the sample population. Thus each analysiswill require two ribozymes, two substrates and one unknown sample whichwill be combined into six reactions. The presence of cleavage productswill be determined using an RNAse protection assay so that full-lengthand cleavage fragments of each RNA can be analyzed in one lane of apolyacrylamide gel. It is not absolutely required to quantify theresults to gain insight into the expression of mutant RNAs and putativerisk of the desired phenotypic changes in target cells. The expressionof mRNA whose protein product is implicated in the development of thephenotype is adequate to establish risk. If probes of comparablespecific activity are used for both transcripts, then a qualitativecomparison of RNA levels will be adequate and will decrease the cost ofthe initial diagnosis. Higher mutant form to wild-type ratios will becorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

Other embodiments are within the following claims. TABLE ICharacteristics of Ribozymes Group I Introns Size: ˜150 to >1000nucleotides. Requires a U in the target sequence immediately 5′ of thecleavage site. Binds 4-6 nucleotides at 5′ side of cleavage site. Over75 known members of this class. Found in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue- green algae, andothers. RNase P RNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portionof a ribonucleoprotein enzyme. Cleaves tRNA precursors to form maturetRNA. Roughly 10 known members of this group all are bacterial inorigin. Hammerhead (HH) Ribozyme Size: ˜13 to 40 nucleotides. Requiresthe target sequence UH immediately 5′ of the cleavage site. Binds avariable number of nucleotides on both sides of the cleavage site. 14known members of this class. Found in a number of plant pathogens(virusoids) that use RNA as the infectious agent (FIG. 1 and 2). Hairpin(HP) Ribozyme Size: ˜50 nucleotides. Prefers the target sequence GUCimmediately 3′ of the cleavage site. Binds 4-6 nucleotides at 5′ side ofthe cleavage site and a variable number to the 3′ side of the cleavagesite. Only 3 known member of this class. Found in three plant pathogen(satellite RNAs of the tobacco ringspot virus, arabis mosaic virus andchicory yellow mottle virus) which uses RNA as the infectious agent(FIG. 3). Hepatitis Delta Virus (HDV) Ribozyme Size: 50-60 nucleotides(at present). Sequence requirements not fully determined. Binding sitesand structural requirements not fully determined, although no sequences5′ of cleavage site are required. Only 1 known member of this class.Found in human HDV (FIG. 4). Neurospora VS RNA (VS) Ribozyme Size: ˜144nucleotides (at present) Cleavage of target RNAs recently demonstrated.Sequence requirements not fully determined. Binding sites and structuralrequirements not fully determined. Only 1 known member of this class.Found in Neurospora VS RNA (FIG. 5).

TABLE II Hammerhead Ribozyme Modifications at the 5′- Terminus orPositions 2.1-2.7 Chemical Composition Ribozyme 5′-Terminus Positions2.1-2.7 U4/U7 Rz 1 OH 2′-ribose 2′-NH₂ Rz 2 OH 2′-deoxyribose 2′-NH₂ Rz3 OH 2′-O—Me 2′-NH₂ Rz 4 OH 2′-O—Me, (2.3-2.7 2′-NH₂ P═S) Rz 5(inactive)* OH 2′-O—Me, (2.3-2.7 2′-NH₂ P═S) Rz 6 NH₂ 2′-O—Me 2′-NH₂ Rz7 (inactive) NH₂ 2′-O—Me 2′-NH₂ Rz 8 NH₂ 2′-O—Me 2′-C-allyl/O—Me Rz 9(inactive) NH₂ 2′-O—Me 2′-C-allyl/O—Me Rz 10 OH 2′-O—Me, (2.3-2.72′-C-allyl/O—Me P═S) Rz 11 (inactive) OH 2′-O—Me, (2.3-2.72′-C-allyl/O—Me P═S)*Catalytically inactive ribozyme cores were produced by substituting2′-O—Me U at positions G5 and A 14.

TABLE III Comparative Catalytic Activities for U4/U7-amino-Containing-Hammerhead Ribozymes k_(obs) (min⁻¹) k_(obs) (min⁻¹) Ribozyme[Rz] = 40 nM [Rz] = 500 nM* Rz 1 0.128 ± 0.032 0.140 ± 0.015 Rz 2 0.019± 0.002 0.023 ± 0.002 Rz 3 0.163 ± 0.012 0.200 ± 0.015 Rz 4 0.108 ±0.001 0.150 ± 0.003 Rz 6 0.131 ± 0.007 0.149 ± 0.007*Neither U4-C-allyl containing ribozymes nor ribozymes containinginactivating nucleotide changes exhibited measurable activity under thestandard conditions employed for these measurements. k_(obs) is derivedfrom two# independent assays and is expressed as average ± range. Values inparentheses express the cleavage rate as a percentage of the controlcleavage rate using Rz 3 at equivalent concentrations.

TABLE IV Inhibition of Rat Smooth Cell Proliferation in Culture RelativeProliferation Index [Ribozyme] nM Ribozyme 50 nM 100 nM 200 nM Rz 1  83± 9 75 ± 12 57 ± 10 Rz 2 104 ± 5 80 ± 6 58 ± 7 Rz 3 103 ± 7 82 ± 13 57 ±10 Rz 4  82 ± 11 31 ± 11 24 ± 5 Rz 5  83 ± 5 31 ± 8 38 ± 5 Rz 6  88 ± 724 ± 7 18 ± 6 Rz 7 104 ± 3 69 ± 13 40 ± 6 Rz 8 106 ± 3 71 ± 9 47 ± 7 Rz9 103 ± 6 87 ± 7 56 ± 7 Rz 10  79 ± 9 17 ± 5 26 ± 12 Rz 11  93 ± 12 69 ±11 32 ± 9Values given represent the percentage of proliferating cell nucleirelative to stimulated lipid-treated cell controls. Mean values of atleast 9 experimental points were used to obtain the relativeproliferative index for each treatment# protocol. Numbers in parentheses represent the standard deviation ofthe mean values. Unstimulated control values were 5 (±2)%. Thepercentage of proliferating nuclei in the serum stimulated control wellswas 72 (±6)%.

TABLE V RNA Synthesis Cycle (2.5 μmol Scale) Wait Reagent EquivalentsAmount Time* Phosphoramidites 6.5  163 μL 2.5 S-Ethyl Tetrazole 23.8 238 μL 2.5 Acetic Anhydride 100  233 μL  5 sec N-Methyl Imidazole 186 233 μL  5 sec TCA 83.2 1.73 mL 21 sec Iodine 8.0 1.18 mL 45 secAcetonitrile NA 6.67 mL NA*Wait time does not include contact time during delivery.

TABLE VI 5′-Amino-5′-deoxynucleotide incorporation Coupling Coupling ofDesilylating Crude Experiment of 5 2′-O-Me-G Reagent AU % FLP 4298 600 s600 s HF/TEA 355.3 14.8 4298 600 s 600 s TBAF 387.3 22.2 4523 600 s 900s TBAF 401.7 21.4 4545 600 s 450 s TBAF 447.8 23.8 4649 300 s 300 s TBAF455.4 27.3

1. A nucleic acid molecule comprising a 5′-cap structure, a 3′-capstructure, or both a 5′- and a 3′-cap structure, wherein said 5′-capstructure is selected from the group consisting of 4′,5′-methylenenucleotide; 1-(β-D-erythrofuranosyl) nucleotide; 1,3-diamino-2-propylphosphate, 3-aminopropyl phosphate; 12-aminododecyl phosphate;hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide;3,5-dihydroxypentyl nucleotide; and 5′-mercapto moieties, and whereinsaid 3′-cap structure is selected from the group consisting of1,5-anhydrohexitol nucleotide; L-nucleotide; threo-pentofuranosylnucleotide; acyclic 3′,4′-seco nucleotide; 3,5-dihydroxypentylnucleotide; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasicmoiety; and 1,4-butanediol, wherein said nucleic acid molecule is singlestranded.
 2. The nucleic acid molecule of claim 1, wherein said nucleicacid molecule is in an enzymatic nucleic acid molecule.
 3. The nucleicacid molecule of claim 1, wherein said nucleic acid molecule is in anantisense nucleic acid molecule.
 4. The nucleic acid molecule of claim2, wherein said enzymatic nucleic acid molecule is in a hairpin,hepatitis delta virus, group I intron, VS RNA or Rnase P RNA motif. 5.The nucleic acid molecule of claim 1, wherein said nucleic acid moleculecomprises said 5′-cap structure.
 6. The nucleic acid molecule of claim1, wherein said nucleic acid molecule comprises said 3′-cap structure.7. The nucleic acid molecule of claim 1, wherein said nucleic acidmolecule comprises said 5′-cap structure and said 3′-cap structure. 8.The nucleic acid molecule of claim 1, wherein said 5′- and 3′-capstructures are different.
 9. The nucleic acid molecule of claim 1,wherein said 5′- and 3′-cap structures are same.
 10. The nucleic acid ofclaim 1, wherein said 3′-cap structure is a 3′-2′ linked invertednucleotide.
 11. The nucleic acid of claim 1, wherein said 3′-capstructure is a 3′-32 linked inverted abasic moiety.
 12. The nucleic acidof claim 1, wherein said 5′-cap structure is a 1,3-diamino-2-propylphosphate group.
 13. The nucleic acid of claim 1, wherein said 5′-capstructure is a L-nucleotide.
 14. The nucleic acid of claim 1, whereinsaid 5′-cap structure is a threo-pentafuranosyl group.
 15. The nucleicacid of claim 1, wherein said 5′-cap structure is a 3,5-dihydroxypentylnucleotide.
 16. The nucleic acid of claim 1, wherein said 5′-capstructure is a 1-(β-D-erythrofuranosyl) nucleotide.
 17. The nucleic acidof claim 1, wherein said 3′-cap structure is a L-nucleotide.
 18. Thenucleic acid of claim 1, wherein said 3′-cap structure is a3,5-dihydroxypentyl nucletide.
 19. An isolated mammalian cell comprisingthe nucleic acid molecule of claim
 1. 20. The mammalian cell of claim19, wherein said mammalian cell is a human cell.